Turner, Stephen T.

Epinephrine is a sympathomimetic which is derived from phenylalanine and tyrosine. The final step of epinephrine synthesis, the conversion from norepinephrine, relies on phenylethanolamine N‐methyltransferase (PNMT). The beta‐2 adrenergic receptor (ADRB2) binds to epinephrine and has several common functional polymorphisms including a Glycine (Gly) for Arginine (Arg) substitution at amino acid 16. We assessed the effect of the gene‐to‐gene interaction of these proteins on the ventricular response to exercise in healthy humans (n=69, age=29±6yrs, ht=174±10cm, wt=71±13kg). We found that within the 2*1 haplotype of the 5′FR, which has previously demonstrated greater epinephrine synthesis compared to 2*7 haplotype, the Arg16 genotype demonstrates lower stroke volume (SV) than the Gly16 genotype despite similar epinephrine levels and synthesis rates (Arg16= 75±5, Gly16=89±6ml/beat). Similarly, within the 2*7 haplotype of the 5′FR, which has demonstrated attenuated epinephrine synthesis compared to 2*1, the Arg16 genotype demonstrates a smaller SV than the Gly16 genotype (Arg16= 59±3, Gly16=78±6ml/beat). Additionally, the Arg16 and Gly16 subjects within the 2*1 haplotype of PNMT have greater stroke volume than their ADRB2 counterparts of the 2*7 haplotype of PNMT. These data demonstrate an interaction between the genes that encode PNMT and ADRB2 on the ventricular response to exercise.

The Gly allele of the Arg16/Gly β 2 ‐adrenergic receptor polymorphism has been associated with hypertension in a linkage analysis in Rochester, MN. Gly homozygotes have greater forearm β 2 ‐receptor mediated vasodilation than Arg after a normal Na + diet (150 mmol/day), and the difference is mediated by endothelial NO. To test the hypothesis that dietary Na + restriction affects β 2 ‐mediated dilation in these groups, we measured forearm blood flow (FBF, plethysmography) during brachial arterial infusion of isoproterenol (ISO) after 5 days of low dietary Na + (10 mmol/day), in healthy subjects homozygous for Arg (n=15, 8 women, 29 ± 8 yrs, mean ± SD) vs. Gly (n=17, 7 women, 31 ± 7 yrs). Baseline FBF did not differ between groups. ISO caused a significant increase in FBF that was not different based on genotype (P = 0.51, main effect of genotype; P = 0.72, genotype‐by‐ISO‐interaction). For example, the highest response to ISO was 10.3 ± 5.5 in Arg and 12.0 ± 4.4 ml/100 ml forearm vol/min in Gly (P t‐test = 0.33). NO inhibition with L‐NMMA decreased baseline FBF, and significantly blunted the response to ISO, but there was no evidence to suggest the responses were influenced by genotype (P = 0.89, genotype‐by‐ISO‐by‐L‐NMMA interaction). We conclude that greater NO‐mediated β 2 ‐receptor vasodilation in Gly homozygotes is no longer apparent following dietary Na + restriction, suggesting that greater dilator function in Gly homozygotes during normal Na + intake may be a protective vascular mechanism in a population where the Gly allele is associated with hypertension. Support: HL‐63328, GCRC RR‐00585, NCRR K23‐17520.

Pulmonary hypertension is common in patients with heart failure (HF); however, for a given degree of left ventricular dysfunction, the range in pulmonary artery pressures (PAP) is large. Polymorphisms of a number of candidate genes have been implicated in contributing to smooth muscle dysfunction and remodeling of the pulmonary vasculature, including the serotonin transporter (5HTT) gene. Patients with HF may have mild tissue hypoxia, which may alter expression of 5HTT. This study examined the influence of a repeat length polymorphism in the promoter region of the 5HTT gene on differences in PAP in HF patients. Ninety HF patients (age=55±14yrs; LVEF=28±10%; NYHA=2.0±0.9) and 90 age and gender matched controls (CTL, age=51±15yrs; LVEF=63±7%) were recruited. Heart failure and CTL subjects were divided into 3 genotype groups, SS‐homozygotes for the short variant, LL‐homozygotes for the long variant and LS‐heterozygous condition. For both HF and CTL, SS n=20; LS n=41; LL n=29). PAP was calculated from the tricuspid regurgitant velocity. Age and gender did not differ between HF and CTL or among genotype groups. PAP was higher in HF compared to CTL (38.9±12.5 vs 27.7±8.2 mmHg, p<0.01, respectively) with a significant genotype effect in HF (SS=34.2±8.6 vs LS=38.2±11.4 vs LL=43.2±15.2 mmHg, p<0.05), but not in CTL. The HF group also demonstrated elevated left atrial diameter compared to CTL (HF 48.0±8.7 vs 32.8±4.8 mm, p<0.01, respectively) although no difference was seen within the HF group across genotypes. These results suggest that the LL variant of 5HTT is associated with elevated PAP in HF but not CTL. HL71478 and HL07111.

Dietary sodium loading decreases β‐adrenergic receptor mediated vasodilation in hypertensive men, and paradoxically increases this dilation in normotensive men; no information exists in women. The purpose of this investigation was to test the hypothesis that dietary sodium loading and restriction influences β‐receptor mediated vasodilation in a sex‐dependent manner. Healthy normotensive subjects consumed a high (400 mmol/day, n=36, mean age±SEM 23±1 yr, 23 women) or low (10 mmol/day, n=30, 30±1 yr, 14 women) sodium diet for 5 days prior to study. Forearm blood flow (FBF, plethysmography) responses to brachial artery administration of isoproterenol (ISO: 1, 3, 6, 12 ng/100 ml tissue/min) were measured before and after endothelial NO synthase inhibition with N G ‐monomethyl‐L‐arginine (L‐NMMA). Consistent with prior reports, the FBF response to ISO was greater in men on high sodium compared to men on low sodium (p=0.03, ISO‐by‐sodium interaction). The highest response to ISO was 16.6±1.7 and 11.0±1.1 ml/100 ml tissue/min (high vs. low, p=0.01). After LNMMA the men's response to ISO did not differ based on sodium. In contrast, in women dietary sodium did not affect the response to ISO before or after LNMMA. We conclude that dietary sodium alters β‐adrenergic receptor mediated vasodilation in men, which appears to be dependent on NO. This is the first study to suggest that these mechanisms of vasodilation are resistant to dietary sodium in women. Support: HL‐089331, CTSA RR‐024150

β 2 ‐adrenergic receptor (ADRB2) gene polymorphisms influence vasodilation, but studies investigating regional or systemic responses conflict, possibly due to counter‐regulatory baroreflex action during systemic β 2 ‐agonist infusion. We tested the hypothesis that ADRB2 gene variation would influence the SVR response to ADRB2 agonist terbutaline (TERB) during baroreflex inhibition. 40 healthy young adults were recruited according to the double homozygous haplotypes: Arg16+Gln27 (n=13), Gly16+Gln27 (n=6), and Gly16+Glu27 (n=21). Arterial pressure was measured by brachial arterial catheter, cardiac output by acetylene breathing. Lymphocytes were sampled for ex vivo analysis of ADRB2 density and binding conformation. Following baroreflex ablation with trimethaphan (3–7 mg/min), phenylephrine was titrated to restore blood pressure to baseline. TERB was infused i.v. at 33 and 67 ng/kg/min for 15 min/dose. There was partial evidence to suggest a main effect of haplotype on the change in SVR ( P = 0.06). For SNP position 16, the highest dose of TERB produced lower SVR in Gly16 vs Arg16 ( P = 0.03). Lymphocyte ADRB2 binding conformation was similar but receptor density was greater in Gly16 vs Arg16 ( P = 0.05). During baroreflex inhibition, the SVR response to systemic ADRB2 agonist is suggestive of augmented ADRB2 function in Gly16+Glu27 homozygotes, with greater influence from Gly16. Supported from NIH and DFG.

Introduction: Thiazide diuretics (TZDs) are the second most frequently prescribed class of antihypertensives. But <50% patients achieve goal blood pressure (BP) on TZD monotherapy, mostly because therapy is chosen overlooking the heterogeneity in patient responses. Circulating microRNAs (miRNAs) remain attractive as non-invasive biomarkers to predict drug response and tailor therapy for optimal outcomes. Hypothesis: Plasma miRNAs may play a role in TZD response; we aim to identify those associated with hydrochlorothiazide (HCTZ) response. Methods: We profiled 754 miRNAs in baseline (untreated) plasma samples of 36 uncomplicated hypertensive adults (18 Responders (R) and 18 Non-responders (NR) based on their diastolic BP (DBP) response to HCTZ therapy) from the Pharmacogenomic Evaluation of Antihypertensive Responses clinical trial, using the real-time qPCR based TaqMan OpenArray Human microRNA panel. We filtered out miRNAs with low amplification scores and Cq>38 and samples with low miRNA expression. Combination of miR-223 and miR-19b (identified as most stably expressed miRNAs by NormFinder Software) was used to normalize expression across samples. We used MannWhitney U test to identify microRNAs differentially expressed between R and NR; linear and logistic regressions for associations of miRNA expression with BP response. FDA corrected p<0.05 was used as significance threshold, unadjusted p<0.05 as suggestive threshold. Results: Mean systolic BP (SBP)/DBP change in R was 14/10 mmHg and in NR 0/1 mmHg. We found 77 miRNAs to be consistently expressed across samples, of which 11 were upregulated and 14 downregulated with >2-fold difference between R and NR. Though no miRNAs reached FDR p<0.05, miR-376a (fold change(fc)= 10.1, p= 0.035) and miR-17 (fc= -0.45, p= 0.017) were differentially expressed between R and NR and their expression associated with DBP and SBP change, along with miR-10b, miR-24, miR-134 associated with SBP change at p<0.05. Conclusions: In this first ever genome-wide miRNA study of antihypertensive drug response, we discovered circulating miRNAs associated with BP response to HCTZ. Future studies are planned to validate these findings in a larger cohort, across different race group and across different TZD.

Objective: Vasopressin increases water permeability and activity of the epithelial sodium channel ENaC in the distal nephron of the kidney, favoring increased water and sodium retention. We hypothesized that genetically mediated differences in circulating vasopressin concentrations could contribute to features of hypertension that vary between non-Hispanic blacks and whites. To test this hypothesis we measured copeptin, a fragment of pre-pro arginine vasopressin with a prolonged half-life that is more easily quantified. Methods: The Genetic Epidemiology of Responses to Antihypertensives (GERA) I study was designed to identify predictors of response to diuretic therapy in non-Hispanic black and non-Hispanic white adults with essential hypertension. Blood samples were obtained after a 4 week washout of anti-hypertensive medication while on a sodium intake of 2 mmol/kg. Copeptin was measured from thawed EDTA plasma samples using the copeptin proAVP homogeneous automated immunofluorescent assay on the BRAHMS Kryptor Compact PLUS (Thermo Fisher). Results: Copeptin was significantly higher in blacks (median: 7.4 pmol/L, IQR: 4.4-10.2 pmol/L) than in whites (median: 5.4 pmol/L, IQR: 3.6-8.0 pmol/L) (p <0.001), while renin was lower (Table 1). Racial differences remained significant (p = 0.001) in multivariate models that included 24 hour urine sodium excretion and calculated plasma osmolality. Conclusions: Higher copeptin (vasopressin) concentrations in non-Hispanic blacks compared to whites may contribute to the distinct differences in the hypertensive phenotypes between these racial groups. The underlying mechanism(s) require further study.

Abstract Acute renal depletion of sorting nexin 1 (SNX1) in mice results in blunted natriuretic response and hypertension due to impaired dopamine D 5 receptor (D 5 R) activity. We elucidated the molecular mechanisms for these phenotypes in Snx1 −/− mice. These mice had increased renal expressions of angiotensin II type 1 receptor (AT 1 R), NADPH oxidase (NOX) subunits, D 5 R, and NaCl cotransporter. Basal reactive oxygen species (ROS), NOX activity, and blood pressure (BP) were also higher in Snx1 ‐/‐ mice, which were normalized by apocynin, a drug that prevents NOX assembly. Renal proximal tubule (RPT) cells from hypertensive (HT) Euro‐American males had deficient SNX1 activity, impaired D 5 R endocytosis, and increased ROS compared with cells from normotensive (NT) Euro‐American males. siRNA‐mediated depletion of SNX1 in RPT cells from NT subjects led to a blunting of D 5 R agonist‐induced increase in cAMP production and decrease in Na + transport, effects that were normalized by over‐expression of SNX1. Among HT African‐Americans, three of the 12 single nucleotide polymorphisms interrogated for the SNX1 gene were associated with a decrease in systolic BP in response to hydrochlorothiazide (HCTZ). The results illustrate a new paradigm for the development of hypertension and imply that the trafficking protein SNX1 may be a crucial determinant for hypertension and response to antihypertensive therapy.