A high performance liquid chromatography (HPLC) method is described for the separation of angiotensin (Ang) peptides and their subsequent quantification by radioimmunoassay in plasma and cerebrospinal fluid (CSF). The use of the ion-pair solvent heptafluorobutyric acid in gradient HPLC achieves baseline resolution of Ang I, Ang II, and the C-terminal fragments des-[Asp1]-Ang I, des-[Asp1]-Ang II, des-[Asp1,Arg2]-Ang II and des-[Asp1,Arg2,Val3]-Ang II in approximately 25 min. Recovery of synthetic Ang standards after phenylsilica extraction and HPLC separation was greater than 70% for each peptide in both plasma and CSF. Ang I and Ang II were shown to be the major immunoreactive Ang components in plasma, and Ang II, des-[Asp1,Arg2]-Ang II and des-[Asp1,Arg2,Val3]-Ang II in CSF.

Angiotensins↗

PubMedDate not supplied

Immunocytochemical localization of angiotensin-(1-7) in the rat forebrain.

Recent findings by this group have led us to reconsider the view that amino (N-) terminal fragments of angiotensin (Ang) II are inactive degradation products of renin-angiotensin system. To further examine this possibility, an antibody to Ang-(1-7), the N-terminal heptapeptide, was produced to demonstrate the neuroanatomical distribution of the rat brain. Ang-(1-7)-immunoreactivity was found in paraventricular, supraoptic, and suprachiasmatic nuclei, bed nucleus of the stria terminalis, substantia innominata, median eminence, and neurohypophysis. This distribution of Ang-(1-7) in the rat forebrain, together with our previous demonstrations of vasopressin secretion in response to this peptide, suggest that Ang-(1-7) functions as a neuromodulator.

Angiotensin I↗

PubMedDate not supplied

Processing of angiotensin peptides by NG108-15 neuroblastoma x glioma hybrid cell line.

The metabolism of angiotensin (Ang) peptides was studied in NG108-15 neuroblastoma x glioma hybrid cells which express Ang II receptors, renin, dipeptidyl carboxypeptidase A (converting enzyme), as well as Ang I and Ang II. In these experiments, 0.2 nM of either 125I-Ang I or 125I-Ang II was incubated with intact cell monolayers and the medium was analyzed for 125I-products by high performance liquid chromatography. The major product generated from the metabolism of labeled Ang I or Ang II was identified as the amino-terminal heptapeptide Ang-(1-7). N-benzyloxycarbonyl-prolyl-prolinal (ZPP), a specific inhibitor of prolyl endopeptidase, inhibited the formation of Ang-(1-7) from Ang I by 35%. Complete inhibition of Ang-(1-7) generation was attained with p-chloromercuriphenyl-sulfonate, which suggests that a sulfhydryl-containing peptidase other than prolyl endopeptidase is also involved in Ang-(1-7) formation. Ang II was observed to be a minor product resulting from Ang I metabolism. Although the converting enzyme inhibitor enalaprilat (MK-422) significantly reduced Ang II formation, it had no effect on the levels of Ang-(1-7). These findings demonstrate a preferential processing of Ang I into Ang-(1-7) which is not dependent on the prior formation of Ang II.

4-Chloromercuribenzenesulfonate↗

PubMedDate not supplied

Reassessment of plasma angiotensins measurement: effects of protease inhibitors and sample handling procedures.

Characterization of C- and N-terminal forms of angiotensin (Ang) peptides mandated assessment of methods to determine plasma levels. 125I-Ang I, 125I-Ang II, and 125I-Ang(1-7) were added to blood samples in the presence of protease inhibitors. Ethylenediaminetetraacetic acid (EDTA) inhibited the conversion of 125I-Ang I to 125I-Ang II. o-Phenanthroline and EDTA (EDTA + o-Ph) did not eliminate [des-Asp1] fragments or 125I-Ang(1-7). The combination of EDTA + o-Ph and pepstatin A or 4-(chloromercuri) benzoic acid (PCMB) significantly reduced 125I-Ang(1-7) generation. Only PCMB plus EDTA + o-Ph eliminated [des-Asp1] fragments. Authentic plasma values of Ang peptides require the correct choice of protease inhibitors.

Angiotensin I↗

PubMedDate not supplied

Circulating neuropeptide Y in dog plasma consists of multiple peptide fragments.

Neuropeptide Y-like immunoreactivity (NPY-LI) in dog plasma was characterized and quantified using three extraction methods (Sep-Pak:acetonitrile, HCl:ethanol, and ethanol). Sep-Pak extraction yields the best recovery and preserves the integrity of the peptide. Oxidized NPY is not generated during blood collection. Using two antisera of different specificities, at least three peptide forms in normal dog arterial and venous plasma were detected. A peptide with retention times similar to oxidized NPY or peptide YY is the major component of plasma NPY-LI under basal conditions, but NPY(1-36) predominates during sympathetic stimulation. The mature peptide in dog plasma is similar to human NPY. The antiserum ABII provides a more accurate measure of circulating NPY(1-36) and its oxidized form. The antiserum ABI is useful for detecting NPY-like fragments.

Animals↗

PubMedDate not supplied

Inhibition of angiotensin converting enzyme by the metalloendopeptidase 3.4.24.15 inhibitor c-phenylpropyl-alanyl-alanyl-phenylalanyl-p-aminobenzoate.

Inhibitors of metallopeptidases may represent new alternatives in the treatment of cardiovascular disease. Recent investigations have linked the hypotensive properties of the metalloendopeptidase 3.4.24.15 (MEP 24.15) inhibitor c-phenylpropyl-alanyl-alanyl-phenylalanyl-para-aminobenzoate (cFP-A-A-F-pAB) to the attenuation of bradykinin metabolism. However, since angiotensin converting enzyme (ACE) is widely recognized to contribute to the metabolic clearance of bradykinin, we characterized the specificity of cFP-A-A-F-pAB towards ACE. We also determined whether cFP-A-A-F-pAB inhibits the conversion of angiotensin I (Ang I) to Ang II by pulmonary ACE. The ACE activity toward the synthetic substrate hippuryl-histidine-leucine (Hip-His-Leu) was measured in vitro using both a purified lung preparation and pooled rat serum. The ACE activity was inhibited at increasing concentrations of the MEP 24.15 inhibitor. Kinetic analysis revealed that cFP-A-A-F-pAB competitively inhibited pulmonary ACE with a Ki of 0.19 microM. In rat serum, cFP-A-A-F-pAB also competitively inhibited ACE. The hydrolysis of Ang I into Ang II by pulmonary ACE was inhibited to a similar extent by both cFP-A-A-F-pAB and the ACE inhibitor MK 422. These findings are the first to show that the MEP 24.15 inhibitor cFP-A-A-F-pAB also inhibits ACE. We suggest that the reported hypotensive actions of cFP-A-A-F-pAB may be due to the reduction in both bradykinin metabolism and Ang II generation arising from the blockade of ACE.

Amino Acid Sequence↗

PubMedDate not supplied

Angiotensin(1-7) in the spontaneously hypertensive rat.

We profiled the concentrations of angiotensin I (Ang I), angiotensin II (Ang II), and angiotensin(1-7) [Ang(1-7)] by the combination of radioimmunoassay and high performance liquid chromatography in the blood of 14-week-old male Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) drinking either tap water or a solution containing ceranapril (30 mg/kg) or lisinopril (20 mg/kg) for 14 days. Differences in the chemical and pharmacokinetic properties of the two converting enzyme inhibitors ruled out class-related effects. Plasma renin activity, angiotensin converting enzyme (ACE) activity, and plasma levels of Ang I and Ang II were the same in vehicle-treated WKY and SHR. In contrast, plasma levels of both Ang(1-7) and vasopressin in SHR were 3.7-fold and 2.6-fold higher, respectively (p < 0.05). Angiotensin converting enzyme inhibition reduced the blood pressure of WKY and SHR, and augmented their intake of water and output of urine. These changes were associated with increases in renin activity and plasma levels of Ang I and Ang(1-7). In both WKY and SHR, lisinopril had a greater effect in inhibiting plasma and cerebrospinal fluid ACE, reducing levels of plasma angiotensinogen, and increasing the concentrations of authentic Ang II. The principal finding of this study is that plasma Ang(1-7) is the sole component of the circulating angiotensin system that is elevated in the established phase of genetic hypertension. The finding that chronic inhibition of ACE augments circulating levels of Ang(1-7) evidenced the existence of functional pathways for the alternate processing of Ang I.

Angiotensin I↗

PubMedDate not supplied

Cardiovascular actions of angiotensin(1-7).

Angiotensin(1-7) had a compound effect on blood pressure of pithed Sprague-Dawley rats. The initial phase of the response consisted of an increase in MAP of short duration and independent of injected dose, followed by a decline of arterial pressure to values below baseline. Both the magnitude (range: -4 +/- 1 to -13 +/- 1 mmHg) and the duration (range: 83 +/- 13 to 255 +/- 17 s) of the depressor response correlated with the dose of peptide. Indomethacin (5 mg/kg) eliminated the depressor component. Only [Sar1,Thr8]Ang II inhibited the effect of Ang(1-7) completely. We conclude that angiotensin(1-7) possesses myotonic actions that are in part related to release of vasodilator prostaglandins through an angiotensin receptor other than AT1 or AT2.

Angiotensin I↗

PubMedDate not supplied

A method to evaluate the elastic behavior of vascular stents.

PURPOSE: To develop a simple method for determining the elastic behavior of vascular stents. MATERIALS AND METHODS: An experimental apparatus was constructed to determine the elastic behavior of four different vascular stents. Each stent was expanded within an artificial compliant vessel and was subjected to an increasing external pressure. The cross-sectional area of each stent was recorded at incremental changes in pressure. Compliance was estimated from the slope of a linear regression analysis fit to the pressure-area data in the elastic range of deformation. RESULTS: The self-expandable stents were the most compliant, and balloon-expandable stents exhibited the least compliance. The balloon- expandable stents initially deformed in an elastic manner and then yielded irrecoverably at higher pressures. CONCLUSION: A simple method has been devised that allows the elastic behavior of stents to be assessed. Quantification of stent compliance with this method is important as a predictor of stent resistance to compression in vivo.

Biomechanical Phenomena↗

PubMedDate not supplied

Effects of vascular stent surface area and hemodynamics on intimal thickening.

PURPOSE: To compare the in vivo response to a new mechanically expandable vascular stent with the response to an existing type of balloon-expandable stent. MATERIALS AND METHODS: Prototype stents were deployed by means of a balloon catheter in the left iliac arteries of four healthy dogs. Palmaz stents were deployed in the contralateral iliac arteries to act as a control, and all stents were explanted after 6 weeks. Arteriography was performed at the time of insertion and before harvest, and pressure gradients were measured across each stent. The stents were then harvested and submitted for histologic examination. RESULTS: The performance of the prototype stent was similar to that of the Palmaz stent with respect to structural integrity, migration, maintenance of intraluminal diameter, ease of deployment, radiopacity, and pressure gradients. Unlike the Palmaz stent, the prototype stent did not foreshorten during expansion. The stents showed a lack of uniformity in terms of the measured luminal area and neointimal accumulation. Neointimal accumulation was more confined to the struts of the prototype stent; the lumen therefore had a fluted appearance. Neointimal accumulation was more broadly distributed around the circumference of the vessel wall of the Palmaz stent. CONCLUSION: In vivo performance of the prototype stent was similar to that of the Palmaz stent. Stent geometry may be an important determinant of neointimal response and resultant long-term patency.

Angiography↗

PubMedDate not supplied

Flow velocity variations in the pulmonary veins of the dog.

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Animals↗

PubMedDate not supplied

Physiological and pharmacological characterization of the area postrema pressor pathways in the normal dog.

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Angiotensin II↗