Zone Sharpening of Peptides in Pressurized Capillary Electrochromatography Using Dynamic pH Junction.

In HPLC, analytes injected into a separation column broaden naturally during the separation procedure. In this paper, analyte zone sharpening of peptides was achieved in pressurized capillary electrochromatography, which is a separation method that combines capillary HPLC and capillary electrophoresis (CE), by employing dynamic pH junction for CE. When the pH of the mobile phase was altered from basic to acidic in a step gradient, the analyte peptides were focused at the basic/acidic interface with the application of voltage. The effect of both pH and pressurized flow velocity on the zone sharpening was investigated. With the proposed method, the peak height of angiotensin II, [Asn(1), Val(5)]-angiotensin II, and angiotensin III were enhanced 12, 10, and 12 times, respectively. Selective peak zone sharpening for angiotensin II was also demonstrated.

[Study on the determination of peptide mixture by HPCE].

A rapid method for the simultaneous assay of 7 peptide mixture, including angiotensin I, II, III, substance P, neurokinin, somatostatin and neurotensin, by high performance capillary electrophoresis has been established. The nature, pH and concentration of buffer, running voltage, detection wavelength, injection time and the effective length of amino-coated capillary were defined with the results of experiment. With 50 mmol/L ammonium acetate (pH 4.5) as running buffer and siphonage injection for 10 seconds, the measurements were carried out at 25 degrees C and 10 kV running voltage [(-)-->(+)] applied to a 57 cm x 75 microns i.d. (50 cm effective length) amino-coated capillary. The 7 peptide mixture was determined by a UV detector at 214 nm. The total time for separation and determination was within 8 min. The recoveries ranged from 95% to 98% with RSD from 2.9% to 4.2%. It has been found that the 75 microns i.d. capillary has higher sensitivity than 50 microns, but its efficiency and Rs were worse.

PC18, a specific aminopeptidase N inhibitor, induces vasopressin release by increasing the half-life of brain angiotensin III.

Angiotensin III (AngIII), which is metabolized in vivo by aminopeptidase N (APN), was previously shown to be one of the main effector peptides of the brain renin-angiotensin system (RAS) in the control of vasopressin release. Recently, a potent APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol, methionine thiol), has been developed. In this study, we first checked the in vitro selectivity of PC18 towards APN, aminopeptidase A (APA) and aminopeptidase B (APB), three zinc metalloproteases with significant identity between their amino acid sequences. The Ki values of this compound on APN were found to be in the nanomolar range (Ki = 8.0 +/- 1.7 nM) but it was 2,150 and 125 times less active on APA and APB, respectively. Secondly, we evaluated in vivo the effect of brain APN inhibition with PC18 on the inactivation of brain AngIII and on vasopressin secretion in mice. For this purpose, mice received [3H]AngII intracerebroventricularly in the presence or absence of the APN inhibitor PC18 (30 microg). At different times after the injection, [3H]AngIII levels were evaluated from hypothalamus homogenates after separation by cation-exchange chromatography. PC18 induced an accumulation of [3H]AngIII, increasing its half-life 3.9 times as compared with control values. In addition, the effect of PC18 on vasopressin release was studied in mice. PC18 (10-100 microgram) was injected intracerebroventricularly, and plasma vasopressin levels were estimated by radioimmunoassay. PC18 increased vasopressin levels in a dose-dependent manner. The maximal increase in vasopressin release (+220%) is observed for a dose of PC18 of 100 microgram and was inhibited 75% by the coadministration of the AngII receptor antagonist (Sar1-Ala8)-AngII (0.5 microgram). These results indicate that in vivo, in the mouse brain, APN inhibition by PC18 increases the half-life of endogenous AngIII, resulting in an enhanced vasopressin release.

Separation of proteins and peptides by capillary electrochromatography in diol- and octadecyl-modified etched capillaries.

This study involves the evaluation of a capillary electrochromatography method based on etching the inner walls of a fused-silica tube, which is subsequently modified by a silanization/hydrosilation reaction scheme. Two different organic moieties, octadecyl and diol, are attached to the etched capillary wall. The performance of these two columns is compared to a bare capillary using peptide (angiotensins) and protein samples. It is concluded that the etching process increases the surface area of the inner wall sufficiently to induce solute-bonded phase interactions for the capillaries modified with the octadecyl and diol moieties. The separation capabilities of the two modified capillaries are not the same, presumably due to differences in the chemical properties of the two ligands. When compared to a bare capillary where separation is due only to electrophoretic mobility effects, the bonded etched capillaries also exhibit significant differences in separation factors for the same solutes under identical experimental conditions.

An immunocytochemical comparison of the angiotensin and vasopressin hypothalamo-neurohypophysial systems in normotensive rats.

In the present study we investigated the possibility that angiotensin II/III and vasopressin coexist in the hypothalamo-neurohypophysial pathway. For our experiments 8-week-old male rats not treated with colchicine were used. The anatomical orientation of the entire pathway for angiotensin and vasopressin was facilitated by examining a series of subsequent coronal, horizontal and sagittal sections. Arching fibre tracts are formed mainly by projections emanating from cell bodies in the paraventricular nucleus, the accessory magnocellular nuclei, the supraoptic nucleus and the retrochiasmatic part of the supraoptic nucleus. The majority extend as far as the median eminence and the neurohypophysis, where major terminal fields exist. However, there is a difference between the staining pattern within the suprachiasmatic nucleus and the hypophysis. The results clearly show the colocalization of angiotensin and vasopressin in neurones as well as in fibres of the hypothalamo-neurohypophysial system.

Separation of radioiodinated angiotensins by chromatofocusing in minicolumns.

Angiotensins I, II, and III (AI, AII, AIII) and Saralasin (Sar1-Ala8-AII) were labeled with 125I and separated from the nonlabeled forms on minicolumns (a Pasteur pipet ) of chromatofocusing medium. At low ionic strength, 125I-labeled angiotensins could be eluted with Polybuffer or a piperazine-histidine buffer at their approximate isoelectric points, while nonlabeled angiotensins remained adsorbed to the column and required 1 mol X liter-1 NaCl for elution. The 125I-labeled angiotensins prepared by this method were bound by antibodies (AI) and adrenal receptors (AII, Saralasin) to an extent similar to angiotensins prepared by DEAE-Sephadex A-25 chromatography. This new method of preparing radioiodinated angiotensins is rapid (15 min), inexpensive, and requires no fraction-collecting equipment.

Angiotensin biosynthesis and concentrations in brain of normotensive and hypertensive rats.

We report here on the extraction and characterization of angiotensin I (ANG I) and angiotensin II (ANG II) from the brain of rats. High pressure liquid chromatography (HPLC) with different mobile phases combined with specific radioimmunoassays (RIA) proved to be a powerful tool for peptide characterization in biological samples; (Ile5)-ANG I, (Ile5)-ANG II and (Ile5)-ANG III could clearly be identified in cerebrospinal fluid (CSF), incubated in vivo and in vitro with renin, in total brain extracts, as well as in hypothalamus (HT), medulla oblongata (MO), cerebellum (CER) and cortex (CO). Angiotensin cleaved from CSF angiotensinogen and angiotensin extracted from brain showed retention times identical to those of plasma angiotensin and synthetic standard peptides, indicating that their amino acid sequence is probably identical. ANG I and ANG II were highest in the HT and lowest in the CO. Following bilateral nephrectomy (NX) both ANG I and ANG II persisted at control levels. Young 10 week old spontaneously hypertensive rats (SHRSP) showed significantly lower ANG I and ANG II concentrations in the HT compared with Wistar Kyoto rats (WKY). Intracerebroventricular (i.c.v.) administration of the converting enzyme inhibitor captopril caused a significant increase in ANG 1 in nephrectomized SHRSP but not in WKY. These differences were not found in 40 week old SHRSP. The data show that ANG I and ANG II are synthetized in the brain of rats. The lower concentrations and the enhanced accumulation of ANG I after converting enzyme blockade in nephrectomized young SHRSP indicate an increased turnover of angiotensin in hypertensive rats.