Measurement of Converting Enzyme Activity by Antibody-Trapping of Generated Angiotensin II: Comparison With Two Other Methods

Activity of the angiotensin converting enzyme (ACE) is usually measured in vitro by estimation of products cleaved by the enzyme from synthetic substrates. These substrates have affinities for ACE different from the natural substrate angiotensin I, and insensitive detection systems necessitate milli-molar substrate concentrations while physiological angiotensin I concentrations are in the picomolar range. A new assay for ACE activity measurement was developed which reliably quantitates femtomoles of generated angiotensin II in plasma from angiotensin I added at a 17 pmol/mL concentration. The production of high affinity monoclonal antibodies against angiotensin II (Kd = 7 × 10-11 mol/L) allowed a quantitative trapping (and thus protection from degrading enzymes) of angiotensin II generated during the incubation step and subsequent ra-dioimmunoassay by simple dilution with labelled angiotensin II. Using 40 µL plasma, the detection limit was 20 fmol/mL/min. Normal human plasma has an ACE activity of 335 ± 83 fmol/mL/min (mean ± SD). Precision was characterized by coefficients of variation of ^ 11% both within-assay and between-assays. Accuracy of the new method was established by comparing ACE activity with the ratio of plasma angiotensin II/angiotensin I in plasma obtained from normal volunteers 0.5 to 24 h after oral administration of 20 mg enalapril. The percentage of ACE inhibition indicated by both methods was almost identical (r = 0.93, n = 60, P < .001). Since the latter ratio appears to reflect in vivo ACE activity, these results indicate that accurate measurement in vitro of ACE activity in vivo has been achieved. Am J Hypertens 1992;5:393-39

Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template

We describe a new rapid and efficient polymerase chain reaction (PCR)-based site-directed mutagenesis method. This procedure is effective with any plasmid and it employs four oligonucleotide primers. One primer contains the desired mutation, the second is oriented in the opposite direction (one of these two primers should be phosphorylated), and the third and fourth should be coding in complementary fashion for a unique restriction site to be introduced in a nonessential region. The method consists of two simultaneous PCR reactions; the PCR products are digested with the enzyme that recognizes the newly introduced unique restriction site and then ligased and used to transform competent bacteria. Additionally, the use of Dpn I facilitates the elimination of template DNA. The newly introduced restriction site is essential for ligation in the correct orientation of the two-PCR products and is further used for mutant screening. Resulting plasmids carry both the new restriction site and the desired mutation. Using this method, more than 20 mutants have already been generated (using two different kinds of templates); all these mutants were sequenced for the desired mutation and transfected into AtT-20 cells and the expressed mutant proteins encoded by the vector were assayed

Effects of Aging and Hypertension on Plasma Angiotensin II and Platelet Angiotensin II Receptor Density

Plasma renin activity (PRA) declines with age in normal individuals, but the effect of age on plasma angiotensin II (ANG II) is less clear. A decline in plasma ANG II with age could result in altered platelet ANG II receptor density since plasma hormone levels influence their target organ receptors. To investigate this possibility, PRA, plasma ANG II, and platelet ANG II receptor density were examined in 17 young, 12 middle-aged, and 14 elderly healthy normotensive volunteers. To assess whether hypertension altered receptor density, these variables were also examined in 23 hypertensive patients. In normotensives, there was a negative correlation between age and PRA (r = — 0.43, P < .05), no significant change in basal plasma ANG II with age, and a weak positive correlation between age and ANG II receptor density (r = 0.34, P < .05). Multiple regression analysis revealed that the relationship between age and ANG II receptor density was independent of the associated rise in mean arterial arterial pressure with age (P < .05). Platelet ANG II receptor density was not significantly related to PRA or plasma ANG II. ANG II receptor affinity did not change with age. Neither PRA nor ANG II receptor density or affinity differed between hypertensives and normotensives of similar mean age, but plasma ANG II was significantly lower in hypertensives compared with normotensives. We concluded that aging is associated with a decline in supine PRA. The small decrease in plasma ANG II was not significant. Platelet ANG II receptor density increased with age primarily due to a small group of elderly subjects with elevated receptor density. There was no change in ANG II receptor density or affinity in hypertensives despite apparently lower plasma ANG II in these patients. Am J Hypertens 1992;5:687-69

Angiotensinergic innervation of the human right atrium: implications for cardiac reflexes

Background: The right atrium is densely innervated and provides sensory input to important cardio-circulatory reflexes controlling cardiac output and blood pressure. Its angiotensin (Ang) II-expressing innervation may release Ang II as a neuropeptide co-transmitter to modulate reflexes but has not yet been characterized. Methods: Intraoperative surgical biopsies from human right atria (n=7) were immunocytologically stained for Ang II, tyrosine hydroxylase (TH) and synaptophysin (SYN). Tissue angiotensins were extracted and quantified by radioimmunoassay. Results: Angiotensinergic fibers were frequent in epicardial nerves and around vessels with variable TH co-localization (none to >50%/bundle). Fibers were also widely distributed between cardiomyocytes and in the endocardium where they were typically non-varicose, TH/SYN-negative and usually accompanied by varicose catecholaminergic fibers. In the endocardium, some showed large varicosities and were partially TH or SYN-positive. A few endocardial regions showed scattered non-varicose Ang fibers ending directly between endothelial cells. Occasional clusters of thin varicose terminals co-localizing SYN or TH were located underneath, or protruded into, the endothelium. Endocardial density of Ang and TH-positive fibers was 30–300 vs. 200–450/mm 2. Atrial Ang II, III and I concentrations were 67, 16 and 5 fmol/g (median) while Ang IV and V were mostly undetectable. Conclusions: The human right atrium harbours an abundant angiotensinergic innervation and a novel potential source of atrial Ang II. Most peripheral fibers were non-catecholaminergic afferents or preterminal vagal efferents and a minority was presumably sympathetic. Neuronal Ang II release from these fibers may modulate cardiac and circulatory reflexes independently from plasma and tissue Ang II sources

Assessment of angiotensin II receptor blockade in humans using a standardized angiotensin II receptor-binding assay

An in vitro angiotensin II (AngII) receptor-binding assay was developed to monitor the degree of receptor blockade in standardized conditions. This in vitro method was validated by comparing its results with those obtained in vivo with the injection of exogenous AngII and the measurement of the AngII-induced changes in systolic blood pressure. For this purpose, 12 normotensive subjects were enrolled in a double-blind, four-way cross-over study comparing the AngII receptor blockade induced by a single oral dose of losartan (50 mg), valsartan (80 mg), irbesartan (150 mg), and placebo. A significant linear relationship between the two methods was found (r = 0.723, n = 191, P < .001). However, there exists a wide scatter of the in vivo data in the absence of active AngII receptor blockade. Thus, the relationship between the two methods is markedly improved (r = 0.87, n = 47, P < .001) when only measurements done 4 h after administration of the drugs are considered (maximal antagonist activity observed in vivo) suggesting that the two methods are equally effective in assessing the degree of AT-1 receptor blockade, but with a greatly reduced variability in the in vitro assay. In addition, the pharmacokinetic/pharmacodynamic analysis performed with the three antagonists suggest that the AT-1 receptor-binding assay works as a bioassay that integrates the antagonistic property of all active drug components of the plasma. This standardized in vitro-binding assay represents a simple, reproducible, and precise tool to characterize the pharmacodynamic profile of AngII receptor antagonists in human

Cardiovascular Effects of Neuropeptide Y

Neuropeptide Y (NPY) is present in the brain, the adrenal medulla, and peripheral sympathetic nerves. This peptide is released together with catecholamines during sympathoadrenal activation. It possesses direct vasoconstrictor properties that are not dependent on simultaneous adrenergic activation. Moreover, it potentiates the vascular effect of several stimulatory substances and may contribute to the modulation of blood pressure responsiveness under a number of circumstances. NPY may also be indirectly involved in the control of blood pressure through regulating the release of hormones with well-established actions on the cardiovascular system. Am J Hypertens 1988;1:193-19

Effect of Cold Pressor Test on the Internal Diameter of the Radial Artery

The aim of this study was to investigate in normal subjects the effect of a cold pressor test on the caliber of the radial artery, a muscular artery of medium size. The internal diameter of this artery was measured continuously using a recently developed ultrasonic device. Immersion of one hand in ice water for two minutes increased blood pressure from 115/75 ± 3/2 (Mean±SEM) to 136/90 ± 6/2 mm Hg (P <.001) and decreased the internal diameter of the radial artery from 2.82 ± 0.12 to 2.60 ± 0.09 mm ( P <.01). These data therefore indicate that the vasoconstriction induced by the cold pressor test involves not only arterioles, but also medium-size arteries. Am J Hypertens 1989; 2:727-72

R-PEP-27, a Potent Renin Inhibitor, Decreases Plasma Angiotensin II and Blood Pressure in Normal Volunteers

The hemodynamic and humoral effects of the specific human renin inhibitor R-PEP-27 were studied in six normal human subjects on low and high sodium intake diets. An intravenous infusion of R-PEP-27 (0.5 to 16 μg/min/kg body wt) reduced blood pressure in a dose-dependent fashion; the mean arterial blood pressure at the end of the infusion fell from 128 ± 4/83 ± 4 to 119 ± 3/71 ± 3 mm Hg (mean ± SEM) (P < .01) during the low sodium intake diet. R-PEP-27 had no effect on blood pressure during the high sodium intake diet. R-PEP-27 significantly reduced plasma angiotensin II and aldosterone concentrations. The temporal response to R-PEP-27 suggests that it is a shortlived although highly potent competitive inhibitor of renin; this peptide is a valuable and specific physiologic probe of the renin-angiotensin system. Am J Hypertens 1994;7:295-30

Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity

Received 22 July 2012; revised 29 January 2013; accepted 4 March 2013Aims Aldosterone plays a crucial role in cardiovascular disease. ‘Systemic' inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the ‘endothelial' MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis. Methods and results C57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high ‘endogenous' aldosterone) and in ‘exogenous' aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR. Conclusion Obesity-induced endothelial dysfunction depends on the ‘endothelial' MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complication