Differential response of human cardiac fibroblasts to angiotensin I and angiotensin II

The vasoactive peptide angiotensin II (Ang II) has been implicated as a mediator of myocardial fibrosis. We carried out a comparative investigation of the effects of Ang II and its precursor Ang I on collagen metabolism and proliferation in cultured human cardiac fibroblasts. Cardiac fibroblasts responded to both Ang I and Ang II with concentration-dependent increases in collagen synthesis but no proliferation. The stimulatory effect of Ang II was abolished by the AT(1) receptor antagonist losartan but not the AT(2) receptor antagonist PD123319. The response to Ang I was not affected by either antagonist, nor by the angiotensin-converting enzyme (ACE) inhibitor captopril. In conclusion, Both Ang I and Ang II stimulate collagen synthesis of human cardiac fibroblasts, the effect of Ang II occurring via the AT(1) receptor whilst Ang I appears to exert a direct effect through non-Ang II-dependent mechanisms. These results suggest distinct roles for angiotensin peptides in the development of cardiac fibrosis.     

Conversion of renin substrate tetradecapeptide to angiotensin II by rat MAB elastase-2

A new approach for the purification of rat mesenteric arterial bed (MAB) elastase-2 has been developed using the chromogenic substrates N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide to monitor the enzymatic activity during various stages of purification. The purified enzyme was evaluated in the presence of various inhibitors and confirmed to have angiotensin (Ang) II-forming ability. The active site-directed inhibitor acetyl-Ala-Ala-Pro-Leu-chloromethylketone (100 micromol x L(-1)), described for human pancreatic elastase-2, abolished the enzymatic activity, confirming that the enzyme is an elastase-2. Chymostatin (100 micromol x L(-1)), an inhibitor regarded as selective for chymases, also showed a remarkable inhibitory effect (94%), whereas captopril (100 micromol x L(-1)) had no effect at all on the Ang II-forming activity. The Ang II precursor renin substrate tetradecapeptide (RS-14P) was converted into Ang II by the rat MAB elastase-2 with the following kinetic constants: Km = 124 +/- 21 micromol x L(-1); Kcat = 629 min(-1); catalytic efficiency (Kcat /Km) = 5.1 min(-1) micro(mol/L)-1. In conclusion, the strategy for the purification of rat MAB elastase-2 with the chromogenic substrates proved to be simple, rapid, accurate, and highly reproducible; therefore, it can be reliably and conveniently used to routinely purify this enzyme. The kinetic parameters for the formation of Ang II from RS-14P by rat MAB elastase-2 emphasize differences in substrate specificity between this and other Ang II-forming enzymes.     

Conformations of synthetic tetradecapeptide renin substrate and of angiotensin I in aqueous solution.

The properties of aqueous solutions of synthetic renin substrate tetradecapeptide (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser) were examined through electrometric titrations, infrared and circular dichroism spectroscopy, and spectrofluorometry. Titration studies of angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) were also made, whose results indicated a flexible folded conformation similar to that previously proposed for the octapeptide angiotensin II, with a possible additional beta turn at the C terminus. The experimental results of the tetradecapeptide study, associated with Chou and Fasman calculations and with an analysis of structure-activity relationships in renin substrates and competitive inhibitors, led to the proposal of a beta turn involving the His-Pro-Phe-His sequence of the tetradecapeptide. This beta turn would be stabilized by beta-antiparallel interaction between residues 3-4 and 10-12 and by electrostatic attraction between the N-terminal ammonium and C-terminal carboxylate groups and would be destabilized below pH 5 by electrostatic repulsion between His6 and His9. The capacity to assume this conformation is related to structural requirements for renin substrates and competitive inhibitors.     

Spontaneous change of human plasma angiotensin I converting enzyme isoelectric point

The isoelectric point of angiotensin I converting enzyme (ACE) spontaneously changes from 4.3 to 4.6 during purification from human plasma. The spontaneous change in pI corresponds to that occurring with neuraminidase-treated but not with EDTA-treated samples. There is no detectable difference in the molecular weight of, or lectin binding by, the two forms of ACE with different pI's. These data indicate that ACE in the circulation contains a greater amount of sialic acid than purified ACE. The implication is that purified ACE isoenzymes which differ in sialic acid content may not reflect tissue-specific isoenzymes but rather artifacts of purification.     

Duplexed iMALDI for the detection of angiotensin I and angiotensin II

An immuno-Matrix Assisted Laser Desorption/Ionization (iMALDI) method has been developed using anti-IgG beads to capture anti-AngI and anti-AngII antibodies, which are incubated with a ～50μL plasma sample to which known amounts of stable-isotope-labeled AngI and AngII have been added. After a short incubation time, the beads are washed, placed directly on a MALDI target, and analyzed by mass spectrometry (MS) and tandem mass spectrometry (MS/MS). The iMALDI assay developed can detect and quantify angiotensin I (AngI) and angiotensin II (AngII) in human plasma. This assay has a Limit of Detection (LOD) of ～10amol/μL (or ～13pg/mL AngI and ～11pg/mL AngII), at a S/N of 2:1, using only one-tenth of the antibody beads which were incubated with a 50-μL plasma sample. This LOD is within the relevant range of patient samples. Little or no angiotensin generation period is required, resulting in a rapid assay. Correlation coefficients for the standard curves are >0.99, with a linear range of 4-100fmol/μL (5-130ng/mL) and 100-2500amol/μL (106-2614pg/mL) for AngI and AngII, respectively. This duplexed assay can quantify AngI and AngII peptide levels simultaneously, in plasma from normotensive and hypertensive patients. The assay can detect changes in the levels of these peptides over time, which will allow quantitation of plasma renin and ACE activities.     

Extraction and recovery of angiotensin II from plasma

A comparison of the three methods of extracting ¹⁴C-angiotensin II from plasma demonstrates that Peart's charcoal method extracts angiotensin II from plasma well, but recovery is inconsistent. Boucher's Dowex method extracts the radioactive labeled angiotensin II from plasma less efficiently than the charcoal method. However, the per cent of the extracted angiotensin in the final recovery product was greater in the Dowex method. Boucher's method is more complicated and time consuming, and less practical. Exeraction of angiotensin with fuller's carth is both simple and convenient, and gives the most consistent and reproducible results.     

Acute angiotensin II increases plasma F2-isoprostanes in salt-replete human hypertensives

Angiotensin (Ang) II induces oxidative stress in vitro and in animal models of hypertension. We tested the hypothesis that Ang II increases oxidative stress in human hypertension, as assessed by plasma F2-isoprostane concentrations. Plasma F2-isoprostanes, hemodynamic and endocrine parameters were measured at baseline and following a 55 min infusion of 3 ng/kg/min Ang II in 13 normotensive and 13 hypertensive volunteers ingesting a high- (200 mmol/d) or low- (10 mmol/d) sodium diet. Mean arterial pressure (MAP) and body mass index were higher in hypertensive subjects. Ang II infusion increased MAP (p<.001) and plasma aldosterone concentrations (p<.001) and decreased plasma renin activity (p<.001) and renal plasma flow (p<.001) to a similar extent in both groups. Plasma F2-isoprostane concentrations were similar at baseline. There was no effect of Ang II on F2-isoprostane concentrations during low-salt intake in either group (normotensive 51.7 +/- 7.1 to 53.7 +/- 6.5 pg/ml and hypertensive 52.2 +/- 8.2 to 56.2 +/- 10.0 pg/ml; mean +/- SE). During high-salt intake, Ang II increased F2-isoprostane concentrations in the hypertensive group (52.3 +/- 7.2 to 63.2 +/- 10.4 pg/ml, p=0.010) but not in the normotensive group (54.2 +/- 4.4 to 58.9 +/- 6.6 pg/ml, p=0.83). Acute Ang II infusion increases oxidative stress in vivo in hypertensive humans. The renin-angiotensin system may contribute to oxidative stress in human cardiovascular disease.     

Determination of the structure of lipid vesicle-bound angiotensin II and angiotensin I

A mass spectrometry (MS)-based strategy was developed to determine the structure of lipid vesicle-bound angiotensin II (AII) and angiotensin I (AI). It involves hydrogen-deuterium exchange (HDX), chemical modifications (e.g., nitration of tyrosine, acetylation of free amino group), and ladder sequencing. HDX is also combined with tandem mass spectrometry (MS/MS) to provide structural details at individual amino acid residues. It was observed that a major portion of both of these peptide hormones interacts with the phospholipid head groups on the surface of the vesicles and that Tyr residue is embedded in the vesicles. Both peptides have a U-shaped structure in the lipid environment.     

Influence of season on plasma antidiuretic hormone, angiotensin II, aldosterone and plasma renin activity in young volunteers

We investigated seasonal changes in hormonal and thermoregulatory responses. Eight volunteers were subjected to the experiment at four times of the year: around the vernal and autumnal equinoxes, and at the summer and winter solstices at latitude 35° N. Plasma antidiuretic hormone (ADH), angiotensin II (ANG II), aldosterone (ALD) and plasma renin activity (PRA) were analyzed before and after water immersion. Seasonal changes in thermoregulatory responses were assessed by measuring core temperature and sweat rate during immersion of the leg in hot water (at 42°C) for 30 min in a room maintained at 26°C. The concentration of plasma ADH and ALD before water immersion was significantly higher in summer than in other seasons. The concentrations of ANG II and PRA did not show seasonal variations. Changes in tympanic temperature during water immersion showed significant differences between seasons, and were higher in winter than in other seasons. The sweat rate was significantly higher in summer than in other seasons. In summary, ADH and ALD concentrations displayed a seasonal rhythm with marked elevation in summer; this may be a compensative mechanism to prevent dehydration from increased sweat loss during summer due to heat acclimatization.