Angiotensin II Synthesis Studies in Dissociated Brain Cell Cultures

Abstract: The biosynthesis of angiotensin II-like peptide was measured in primary cultured brain cells from the fetal rat. Cells from the whole brains of 20-day gestational age Sprague-Dawley rats were dissociated by mild trypsinization and grown for 5 days in supplemented (serum-free) medium prior to experimental analysis. The time-dependent incorporation of [³H]proline into newly synthesized angiotensin II-like peptide was measured by radioimmunoassay using specific angiotensin II antisera. Analysis of radioimmunoassay data revealed an increase in the amount of tritium-labeled angiotensin II in the crude extract of the brain cells during the first 24 h in culture. The chromatographic character of the angiotensin II-like peptide was further identified by high pressure liquid chromatography. Elution profiles for newly synthesized angiotensin II were identical to those profiles generated for [³H]angiotensin II and nonradiolabeled angiotensin II standards. To determine the bioactivity of the angiotensin II-like peptide, fractions of the column-purified peptide were injected into the region of the rat lateral ventricle via an indwelling cannula. Systolic pressure increased up to 20 mm Hg depending upon the amount of peptide injected. These data clearly support the existence of an endogenous renin-angiotensin system in dissociated brain cell cultures from the fetal rat, and provide an experimental model for further analysis of the regulatory mechanisms of angiotensin II synthesis.     

Biochemical Studies on Polyamine and Its Analogues

The phagocidal action of oxidation products of natural and synthetic amines by crystalline amine oxidase preparations has been investigated with a large variety of bacteriophages. Oxidized spermine was shown to inactivate T3, T5, T7, MS2, ?80, λ (Escherichia coli); ε¹⁵, ε³⁴ (Salmonella anatum); Al (Bacillus subtilis); P465, P468, Ap85, P4 (Brevibacterium lactofermentum); I128T (Pseudomonas glycinea), I2418 (Xanthomonas phaseoli) and PK66 (Streptomyces griseus). Phages of T2, T4, T6, ?X174 (E. coli), P22 (S. anatum) and M2, SP10 (B. subtilis) was not inactivated. Essentially the similar activity was observed with the oxidation products of spermidine and synthetic polyamines analogous to spermine.

The reduction of logarithmic titers of survival phages by the oxidized polyamines proceeded linearly with both incubation time and concentration of the oxidized polyamines. Then, it is conceivable that the inactivation of phages may be due to the interaction between oxidized polyamine and phagal nucleic acid.     

Biochemical Studies on Polyamine and its Analogues

Preincubation with spermine, of λ T 7 and P 465 phages which were sensitive to oxidized spermine, resulted in a decrease of their susceptibility to the action of oxidized spermine. Phages resistant to oxidized spermine such as T 4 and ?X 174 became susceptible to this agent after dialysis.

The mechanism of phagocidal action of oxidized spermine was examined with ³²P-labelled λ phage. Oxidized spermine interfered neither with the absorption of λ phage, nor with the injection of its DNA into the host cells. The injected DNA, however, did not lead to the formation of mature phage.

The interaction of oxidized spermine with the DNA of phages T 4 and T 7 was investigated by thermal denaturation studies. DNA treated with oxidized spermine showed the same Tm as untreated DNA. However, the treated DNA was decreased in its hyperchromicity and was renatured to a great extent, even after rapid cooling. These facts are explained by the formation of cross-links which prevents the separation of complementary DNA strands.     

Angiotensin II and aldosterone regulation

The circulating renin-angiotensin system is a major regulator of the secretion of the adrenocortical hormone, aldosterone. This renin-angiotensin aldosterone system is important in the control of salt and water balance and blood pressure. This review describes the historical background leading to the discovery of aldosterone in the 1950s and the recognition in the 1960s that angiotensin II was involved in its control. Although angiotensin II is important in the regulation of aldosterone secretion, its action is influenced by multiple other factors, especially potassium and atrial natriuretic peptide. In addition to the circulating renin-angiotensin system, a local renin-angiotensin system is present in the zona glomerulosa cell. This local system also appears to be involved in the regulation of aldosterone production. The mechanism by which angiotensin II stimulates the adrenal zona glomerulosa cell is described in some detail. Angiotensin II interacts with the angiotensin receptor (AT1) membrane receptor that is coupled to cellular second messengers. Specific AT1 receptor antagonists are now clinically used to block angiotensin II's action on various target organs, including the adrenal gland.     

Angiotensin II and extracellular matrix homeostasis

As a circulating hormone, endocrine properties of angiotensin (Ang) II are integral to circulatory homeostasis. Produced de novo its autocrine/paracrine properties contribute to biologic responses involving various connective tissues (e.g. extracellular matrix, adipose tissue, bone and its marrow). In this brief review, we develop the concept of extracellular matrix homeostasis, a self regulation of cellular composition and structure, wherein fibroblast-derived AngII regulates elaboration of TGF-beta 1, a fibrogenic cytokine responsible for connective tissue formation at normal and pathologic sites of collagen turnover.     

Angiotensin II receptors: Localization of type I and type II in rat epididymides of different developmental stages

Correspondence to: H.C. Chan--> Abstract. Previous studies from our laboratory have provided evidence for the existence of a local renin-angiotensin system in the rat epididymis. Evidence has also accumulated, indicating that locally formed angiotensin II from the rat epididymis may play a paracrine and/or autocrine role in regulating epididymal electrolyte and fluid transport. In the present study, specific anti-peptide antibodies against the second extracellular loops of angiotensin II type I (AT₁) and type II (AT₂) receptors were used to localize immunocytochemically these receptors in the rat cauda epididymides of three developmental stages, namely, immature (2-week), early mature (6-week) and fully mature (10-week). The immunostaining intensity for AT₁ receptors was found to be stronger than that for AT₂ receptors throughout rat epididymides of all stages. However, the immunostaining for both AT₁ and AT₂ receptors observed in the fully mature rat epididymis was much more intense than that observed in the epididymides of the two younger stages. While the immunostaining for both AT₁ and AT₂ receptors in the younger rat epididymides appeared to be distributed in both basal and apical regions, the immunostaining in the fully mature epididymis was predominantly localized in the basal region. The present finding of the differential patterns of angiotensin II receptor immunoreactivity in three different stages of the rat epididymis may reflect the fine tuning of rat epididymal function by angiotensin II, acting as a paracrine or autocrine agent, during the course of development. Received: 18 December 1995/Revised: 19 December 1996     

心钠素的结构与功能的研究

心钠素的结构与功能的研究王进,施薄涛（中国科学院上海生物化学研究所,２０００３１）关键词心钠素ＡＰＩＩＩ;类似物;舒张;利尿;降血压心钻素（ＡＮＰ,ａｔｒｉａｌｎａｔｒｉｕｒｅｔｉｃＰｅＰｔｉｄｅ）是ＤｅＢｏｌｄ在１９８１年发现的一种由大鼠心肌细胞分...     

Angiotensin I converting enzyme (kininase II) in isolated retinal microvessels.

Swine retinae were homogenized and fractions enriched in retinal microvasculature were prepared by techniques of selective sieving and centrifugation. The identity and purity of the preparations were investigated by phase contrast and electron microscopy. Angiotensin I converting enzyme (kininase II) was concentrated in the retinal microvessels. Metabolism of angiotensins and kinins in localized sites of the vasculature may contribute to local regulation of blood flow.     

Endogenous and Expressed Angiotensin II Receptors on Xenopus Oocytes

Intact Xenopus oocytes contain a homogeneous population of binding sites for the angiotensin II (Ang II) receptor antagonist 125I-[Sarc1,Ile8]-Ang II (125I-SARILE). Binding of 125I-SARILE to intact oocytes was saturable and of high affinity with an apparent KD of 0.7 nM and maximal density of 0.12 fmol/oocyte. Binding of 125I-SARILE to oocytes also was specific for Ang II-related peptides with a rank order potency of: [Sarc1]-Ang II greater than Ang II greater than Ang III much greater than Ang I. However, these endogenous binding sites were present only in follicle-enclosed oocytes and within the follicular layer itself. On the other hand, injection of poly(A)+ RNA isolated from murine N1E-115 neuroblastoma cells into oocytes resulted in the appearance of 125I-SARILE binding sites even in defolliculated oocytes. These expressed receptors exhibited pharmacological properties similar to those endogenously present in the follicular layer, although their levels were much less. Collectively, these results suggest that endogenous Ang II receptors are present on Xenopus oocyte follicle cells, whereas Ang II receptors expressed from exogenous N1E-115 RNA are found on the oocytes themselves. In addition, the high density of Ang II receptors on the follicle cells emphasizes the necessity for care in using Xenopus oocytes for the expression of receptors encoded by exogenous RNAs.     

Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates

Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC⁰-AngI and TOAC¹-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe⁸-His⁹ peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr⁴ fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra.