Aging and Human Hormonal and Pressor Responsiveness to Angiotensin II Infusion With Simultaneous Measurement of Exogenous and Endogenous Angiotensin II

A decline in the function of the renin angiotensin aldosterone system may induce adaptive changes in response to angiotensin II (ANG II) with age. We have examined platelet ANG II receptor density, blood pressure and aldosterone responses to ANG II [Asn1, Val5-ANG II] (Hypertensin, Ciba Geigy, Horsham, Sussex, England) infusion in 8 young, 24 to 30 years, and 8 older, 54 to 65 years, healthy volunteers. To measure circulating ANG II, we established a new method for specific and simultaneous measurement of exogenous [Asn1,Val5] (Hypertensin) and endogenous [Asp1,Ile5] ANG II in plasma by using isocratic HPLC and radioimmunoassays with cross-reacting antibodies and compared results with immunoreactive ANG II which was measured conventionally using monoclonal antibodies. Baseline endogenous ANG II (Asp1,Ile5-ANG II) levels in venous plasma were marginally, but not significantly, lower in the old [mean (95% confidence limits): 3.4 (< 0.1 to 7.7) ν 3.7 (1.2 to 6.2), fmol/ mL] and during suppression by the Hypertensin infusion appeared consistently, but not significantly, lower in the old [0.9 (0 to 3.1) ν 2.1 (0.6 to 3.7), after 3 ng/kg/min], while the same infusion rate in young and old resulted in similar plasma Hypertensin levels. Baseline systolic blood pressure (SBP) was similar in both groups but the percentage increases in SBP at infusion rates of 1, 3.0, and 10 ng/kg/min were greater in the old than in the young (9.1 ν 2.8, Ρ<.05; 16.3 ν 8.0, Ρ<.01; 30.4 ν 14.0%, Ρ<.001, respectively). The increment in diastolic blood pressure or mean arterial pressure during infusion did not differ significantly between the groups. Heart rate fell significantly during ANG II infusion in young (P < .05) but not old subjects. Basal plasma aldosterone was similar in both groups. However, the percentage increases in plasma aldosterone were greater in young than old at infusion rates of 1.0, 3.0, and 10.0 ng/kg/min (94 ν - 5 ; 238 ν 28; 462 ν 69%, all Ρ<.001). Platelet ANG II receptor density was elevated in the old [6.5 (1.9 to 11.1) ν 1.14 (0.21 to 2.07), fmol/109 cells, Ρ<.01]. Our findings demonstrate that it is possible to measure specifically and simultaneously exogenous and endogenous ANG II in plasma during Hypertensin infusion and that endogenous ANG II concentrations decrease with increasing levels of exogenous ANG II. During infusion, similar plasma Hypertensin levels were attained in both young and old, but systolic blood pressure responsiveness was increased in the old, while adrenal responsiveness was reduced. Am J Hypertens 1993;6:641-64

Volume expansion enhances plasma endothelin-1

We hypothesized that acute volume expansion by saline infusion triggers the release of endothelin-1. Bolus intravenous saline infusion (8 mL/min) in six groups of conscious Wistar rats and spontaneously hypertensive rats did not change mean arterial pressure or heart rate (n = 8 to 12). At 1 min after infusion, the plasma endothelin-1 level was significantly increased in Wistar rats and in spontaneously hypertensive rats by 42% and 61%, respectively (unpaired data). In 12 Wistar rats, the endothelin-1 level increased from 0.68 ± 0.13 to 1.19 ± 0.17 fmol/mL (mean ± SEM, P < .0001, paired data). Thus, acute volume load by rapid saline infusion increases plasma endothelin-1 levels. Vasoconstriction induced by endothelin-1 may counteract enhanced circumferential stretch created by volume expansion. Am J Hypertens 2003;16:1057-1061 @ 2003 American Journal of Hypertension, Lt

O-36: The orally active renin inhibitor SPP100 blocks the renin-angiotensin system in humans equally well as enalapril

The activity of the renin-angiotensin system (RAS) is mainly determined by the concentration of active renin. Direct inhibition of renin is therefore a primary goal for blocking the RAS. So far, all specific renin inhibitors lacked potency or were clinically ineffective after oral administration. We tested the new orally active non-peptidic renin inhibitor SPP100 in 18 healthy volunteers on a constant sodium diet (100 mmol/day)using a double-blind, threeway crossover protocol. In 3 periods of 8 days, separated by wash-outs of 1 week, each volunteer received 2 dosage levels of SPP100 once a day (40,80,160 or 640mg) and placebo or 20mg enalapril. SPP100 was well tolerated. Not surprisingly, blood pressure and heart rate remained unchanged in these normal volunteers. SPP100 plasma levels showed that steady state was reached after 8 days of dosing. The table below summarizes median plasma levels at peak (P, 0.5-6h) and trough (T, 24h after dosing)on day 8: In conclusion, the renin inhibitor SPP100 dose-dependently blocks the RAS and decreases angiotensin levels in human subjects following oral administration. The effect is long-lasting and at 160mg at least equivalent to that of 20mg enalapril. SPP100 has the clear potential to become the first renin inhibitor that provides a true alternative to ACE-inhibitors and Ang II receptor antagonists in the therapy of hypertension, cardiovascular and renal disease. Placebo SPP 100 Enalapril 40mg 80mg 160mg 640mg 20mg Renin pg/ml P 12 34 95 130 670 330 T 11 19 39 64 373 58 PRA ng/ml/h P 1.0 0.28 0.21 0.16 0.08 27 T 1.4 0.89 0.60 0.41 0.39 3.5 Ang I fmol/ml P 3.2 1.6 1.4 0.33 0.70 350 T 7.0 7.1 6.1 4.8 3.1 34 Ang II fmol/ml P 3.0 1.5 1.5 0.61 0.27 0.88 T 4.5 3.7 3.5 3.2 2.5 4.

OR-53: Plasma immunoreactive endothelin-1 levels in hypertensive rats and human subjects

Endothelin-1 (ET-1) is an endothelium-derived potent vasoconstrictor peptide of 21 amino acids. To establish reference values in different forms and models of hypertension and in human subjects an assay for plasma ET-1 was optimized. Immunoreactive (ir-) ET-1 is extracted by acetone from 1 ml plasma and subjected to a sensitive sandwich type enzyme linked immunosorbent assay. The detection limit for plasma ET-1 (3 SD above zero readings) is 0.05 fmol/ml. Mean recoveries of the 1, 2, 5, 10 fmol of ET-1 added to 1 ml plasma (n = 5, each) were 66, 75, 85 and 92 % respectively. The within-assay coefficients of variation were 12, 5, 3, 3 and 0.5 % for plasma ET-1 concentrations of 0.84, 1.5, 2.3, 5.2 and 9.9 fmol/ml respectively. Between-assay coefficients of variation for two human control plasmas containing 1.0 fmol/ml (n = 8) and 1.2 fmol/ml ET-1 (n = 7) were 8% and 10% respectively. Assay accuracy was demonstrated by the consistent recoveries of added ET-1 and by the linearity of ir-ET-1 concentrations measured in serially diluted plasma extracts (r = 0.99). No ir-ET-1 was detected when albumin buffer was extracted instead of plasma (buffer blank). Using this method, we found increased ir-ET-1 levels in plasma of three experimental rat models of hypertension. (i) Plasma ir-ET-1 concentrations were significantly higher in stroke-prone spontaneously hypertensive rats (SP-SHR) than in normotensive Wistar rats. (ii) DOCA-salt hypertensive rats exhibited 4 times higher ir-ET-1 levels than sham operated control rats. (iii) One kidney-one clip (1K-1C) hypertensive rats showed moderately increased ir-ET-1 levels compared to sham operated controls. In contrast, the ir-ET-1 levels in plasma of SHR were half that of normotensive Wistar rats. In two kidney-one clip (2K-1C) Goldblatt hypertensive rats, the plasma ir-ET-1 concentrations were not different from sham-operated control rats. The plasma ir-ET-1 concentrations of 37 healthy human subjects were 0.85 ± 0.26 fmol/ml (mean ± SD). We conclude that the present assay reliably measures plasma immunoreactive ET-1 levels in rats and in human subjects. Normal plasma ET-1 concentrations in humans and conscious rats are in the low picomolar rang

OR 47: Renal endothelin receptor type B upregulation in rats with low or high renin hypertension

Endothelin-1 (ET-1) is a potent renal and systemic vasoactive peptide. It acts through ETA and ETB receptors. We investigated density and subtype distribution of ET-1 receptors in hearts and kidneys of normotensive and hypertensive rats. Five groups of uninephrectomized Wistar rats were put on a low salt diet for six weeks. During this period, three groups of rats drank tap water and two groups received saline. One group of each regimen received DOCA subcutaneousely (1.6 mg/day). The fifth group of rats had the left renal artery clipped to induce 1K1C hypertension. At 6 weeks, mean arterial pressure (MAP) was recorded in conscious rats via a femoral artery catheter. Binding assays using 125I-ET-1 were carried out on membrane preparations in the presence and absence of the ETA receptor antagonist FR139317. On tap water, MAP was at 121.8±3.3 mmHg and DOCA or saline did not raise this MAP. On DOCA-salt and in 1K1C rats, MAP was increased to 154.5±5.8 mmHg (p<0.001) and 218.4±10.5 (p<0.001) mmHg, respectively. ET receptor subtypes were not equally expressed in the heart and the kidney: ETA was predominantly expressed in the heart, whereas ETB was predominant in the kidney. Both hypertensive models, the DOCA-salt and the 1K1C rats showed further significant changes: i) Cardiac weight index compared to controls of 2.49±0.06 mg/g was higher (p<0.001) at 3.89±0.10 and 4.86±0.18 mg/g in DOCA-salt and 1K1C hypertension, respectively, and kidney weight index compared to controls of 4.78±0.22 mg/g was higher at 10.10±0.54 mg/g in DOCA-salt (p<0.001) but tended to be below controls in 1K1C rats. ii) In the kidneys, the density of the ETB receptor subtype was upregulated in DOCA-salt and 1K1C rats from 160±8 to 217±12 and 190±2 fmol/mg (p<0.05), respectively, and ETA tended to be downregulated. iii) Plasma renin activity was decrased in DOCA-salt rats from 17±3 to 0.17±0.01 ng/ml/h and increased in 1K1C rats on low salt diet to 30±5 ng/ml/h (p<0.001). We conclude that upregulation of the ETB receptor mediating vasodilation and downregulation of the ETA receptor mediating vasoconstriction is compatible with a mainly renal counterregulatory effect of endothelin-1 to hypertension. This counterregulation may occur in both low and high renin models of hypertension. Am J Hypertens (2004) 17, 20A-21A; doi: 10.1016/j.amjhyper.2004.03.04

P-537: Pioglitazone blunts the blood pressure response to angiotensin II in insulin-resistant zucker rats

Thiazolidinediones are high-affinity agonists of peroxisome proliferator-activated receptors (PPAR)-γ which have been found to lower blood pressure (BP) in animal models of diabetes and reno-vascular hypertension. The mechanisms leading to this decrease in blood pressure are still unclear. Since the renin-angiotensin system is a main regulator of blood pressure, we investigated the effects of pioglitazone on blood pressure, plasma renin activity and on the blood pressure response to exogenous angiotensin II in insulin-resistant Zucker rats. Pioglitazone 20mg/kg/d or vehicle were administered for 4 weeks to 8-weeks old fa/fa Zucker rats. Pioglitazone-treated rats were heavier than vehicle-treated rats (respectively 481g±8g vs 437±5g, p=0.0002, mean±SEM) and ate more (35.6±0.5g/d vs 28.9±0.3, p<0.0001). The increase in blood sugar after an intra-venous glucose tolerance test was significantly attenuated in the pioglitazone treated rats at 10,15 and 30 minutes. Systolic (SBP), diastolic (DBP) blood pressure and heart rate (HR) were lower in pioglitazone-treated rats: SBP: 124±3 mmHg vs 144±3, p<0.001; DBP: 80±2 vs 94±2, p<0.001; HR: 369±6 vs 397±8, p<0.01. The BP response to exogenous angiotensin II was significantly attenuated in pioglitazone treated rats. With the 25 ng/kg Ang II dose the increase in BP was 27.8±2.4 mmHg with pioglitazone and 37.5±3.3 with the vehicle (p=0.04) and with the Ang II 100 ng/kg dose the increase in BP was respectively 36.1±2.7 mmHg and 49.2±2.3 (p=0.003). Plasma renin activity was comparable in both groups. In conclusion, these results show that pioglitazone blunts the BP response to angiotensin II in insulin-resistant Zucker rats. This effect may partially explain the blood pressure lowering effect of PPAR-γ agonist

P-620: Pioglitazone stimulates renin and favors sodium retention and weight gain in healthy subjects

Glitazones induce peripheral edema through an unknown mechanism in up to 20% of cases. This study examines the effects of pioglitazone (PIO) on renal sodium handling and renal hemodynamics in healthy male volunteers (HV) exposed to a high (HS) and low (LS) sodium diet. The influence of PIO on plasma renin activity (PRA), aldosterone and atrial natriuretic peptide (ANP) was examined. Nine HV aged 22-28 y were enrolled. BMI, blood pressure and glucose tolerance were normal. The study had a double-blind, randomized, placebo controlled, twofold cross-over design. Each subject received either PIO 45 mg qd or placebo qd, for 6 weeks, with 2 weeks wash-out. From weeks 1-4, subjects were on their usual diet. During weeks 5 and 6, subjects were either on a LS or a HS diet for a week which was followed by ambulatory blood pressure measurements, hormonal measurements and renal function studies. The differences between PIO and placebo effects were examined (median, range among all subjects). No subject developed edema. Insulin sensitivity, systolic and diastolic blood pressure, glomerular filtration rate, renal plasma flow or filtration fraction did not change significantly with PIO. Weight increased with PIO in 7/9 subjects while on a LS diet (0.7kg; -1-2.9) and in 6/9 while on a HS diet (1.1kg; -1.5-3.4). Median sodium (Na) excretion with placebo was of 21.2mmol/24h and 239.7mmol/24h respectively while on a LS and HS diet. Urinary Na excretion decreased with PIO in 6/9 subjects on a LS diet (-12.2mmol/24h; -21-8.7, p=0.05), and in 5/9 subjects on a HS diet (-30mmol/24h; -344-69). Na clearance decreased in 6/9 subjects on a LS diet (-0.05 ml/min; -0.11-0.05) and in 6/9 subjects on a HS diet (-0.15 mmol/ml; -2.7-0.4). Na clearance at the proximal level decreased with PIO in 8/9 subjects on a LS diet (-4.9 ml/min; -14.4-1.5, p=0.01) and in 5/9 subjects on a HS diet (-2 ml/min; -43-8.5). PRA increased with PIO in 8/9 subjects on a LS diet (0.16 ng/ml/h; -0.07-0.8, p=0.02) and on a HS diet (0.09 ng/ml/h; -0.1-0.21, p=0.03). Aldosterone increased in 5/9 subjects on a LS diet (6 pg/ml; -19-144) and in 6/9 subjects on a HS diet (5 pg/ml; -20-74). ANP levels did not change. In conclusion, pioglitazone increases PRA, independently from Na intake and favors Na retention in healthy volunteers. This mechanism could contribute to the development of edema in subjects treated with glitazone

Measurement of plasma endothelin-1 in experimental hypertension and in healthy subjects

Background: Endothelin-1 is an endothelium-derived potent vasoconstrictor peptide of 21 amino acids. To establish reference values in different models of hypertension and in human subjects an assay for plasma immunoreactive endothelin-1 (ET-1) was optimized. Methods: ET-1 is extracted by acetone from 1 mL of plasma and subjected to a sensitive enzyme-linked immunosorbent assay. Results: The detection limit for plasma ET-1 is 0.05 fmol/mL. Mean recoveries of the 1, 2, 5, and 10 fmol of ET-1 added to 1 mL of plasma were 66%, 75%, 85%, and 92%, respectively. Within- and between-assay coefficients of variation were ≤12% and ≤10%, respectively. Assay accuracy was demonstrated by consistent recoveries of added ET-1 over the entire physiologic range of plasma concentrations and by the linearity of ET-1 concentrations measured in serially diluted plasma extracts (r = 0.99). No ET-1 was detected when albumin buffer was extracted instead of plasma. Using this method, we found increased ET-1 levels in plasma of three experimental rat models of hypertension: stroke prone spontaneously hypertensive rats (SP-SHR), deoxycorticosterone acetate-salt hypertensive rats, and one kidney-one clip hypertensive rats. In contrast, plasma ET-1 levels of SHR were half those of normotensive Wistar rats. In two kidney-one clip hypertensive rats, plasma ET-1 concentrations were not different from those found in sham-operated control rats. Plasma ET-1 concentrations of 37 healthy men were 0.85 ± 0.26 fmol/ml (mean ± SD). Conclusions: The present assay reliably measures ET-1 levels in rat and human plasma. It allows to discriminate between different forms of hypertension with high or low circulating levels of ET-1. Am J Hypertens 2003;16: 515-521 @ 2003 American Journal of Hypertension, Lt

P-624: Changes in plasma renin match the antihypertensive effects of aliskiren in patients with hypertension: Placebo/irbesartan-controlled trial with the orally active renin inhibitor aliskiren

For several decades, the lack of oral availability and poor antihypertensive effects of renin inhibitors (RI), despite seemingly powerful inhibition of conventionally measured plasma renin activity (PRA), have discredited RI as cardiovascular drugs. Aliskiren is a novel orally effective RI with antihypertensive potency comparable to losartan or irbesartan. The present study investigated the effects of aliskiren and irbesartan on PRA, measured by the reliable antibody trapping technique, as well as on plasma active renin concentration (ARC) and sitting systolic blood pressure (SBP). In 569 patients with mild to moderate hypertension (baseline sphygmomanometric sitting blood pressure 152±12/99±4 mmHg, mean±SD), PRA and ARC, as well as SBP were measured before and after 8 weeks of treatment with once daily oral doses of aliskiren (150, 300 or 600mg), irbesartan 150mg or placebo. The effects of study treatments on PRA, ARC and SBP are summarized in the Table. Treatment N PRA (ng/mL/h) ARC (pg/mL) SBP (mmHg) Baseline Week 8 Baseline Week 8 Baseline Week 8 Placebo 111 0.72 0.64 6.2 5.6 152 ± 12 147 ± 18 Aliskiren 150mg 112 0.66 0.20 6.0 15.3 151 ± 11 140 ± 14 Aliskiren 300mg 115 0.59 0.17 6.1 21.0 152 ± 10 137 ± 14 Aliskiren 600mg 113 0.64 0.16 5.8 34.9 153 ± 12 137 ± 16 Irbesartan 150mg 118 0.64 1.33 5.5 11.3 153 ± 11 140 ± 16 PRA and ARC values are geometric means; SBP values are mean ± SD Aliskiren reduced PRA by 69%, 71% and 75% at 150, 300 and 600mg respectively, while irbesartan doubled PRA. Most of the antihypertensive effect of aliskiren was obtained with the lowest dose, but higher doses slightly further decreased SBP. Aliskiren 150mg and irbesartan 150mg provided similar increases in ARC and hence comparably blocked the renin-angiotensin system (RAS), and the achieved SBP was also the same. Aliskiren 300mg and 600mg caused greater increases in ARC compared with irbesartan 150mg (p<0.05), and further decreases in SBP. The dose-dependent increases in ARC observed with aliskiren document increasing blockade of the RAS. In conclusion, aliskiren provides a parallel reduction in PRA and SBP, a dose-dependent blockade of the RAS and is at least as effective as irbesartan at comparable dosages (150mg