Quantitative in vitro autoradiographic characterization of [125I]angiotensin III binding sites in rat adrenal gland

A single class of high-affinity binding sites for [125I]angiotensin III and [125I]angiotensin II were found in rat adrenal medulla and zona glomerulosa by quantitative autoradiography. In the medulla, Kd were 1.46 and 1.16 nM, and Bmax 1700 and 1700 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. In the zona glomerulosa, Kd were 0.86 and 0.90 nM, and Bmax 790 and 560 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. Unlabeled angiotensin III and angiotensin II displaced [125I]angiotensin III with similar potency in both adrenal zona glomerulosa and medulla. Our findings suggest that angiotensin III and angiotensin II might share the same binding sites in adrenal gland and support the hypothesis of a role for angiotensin III in the adrenal medulla and zona glomerulosa.     

Enzymatic formation of angiotensins II and III in the hindlimb circulation of dogs

Experiments were performed in 14 anesthetized dogs to (1) to determine if the reductions in hindlimb blood flow produced by [des-Asp1] angiotensin I were due to its local enzymatic (kininase II) conversion to angiotensin III and (2) to quantitate the extent of conversion of angiotensin I to angiotensin II and of [des-Asp1] angiotensin I to angiotensin III in the hindlimb circulation. Graded doses of these peptides were administered as bolus injections directly into the left external iliac artery while measuring flow in this artery electromagnetically. Dose-response relationships were determined before and during the inhibition of kininase II activity with captopril or antagonism of angiotensin receptor sites with [Ile7] angiotensin III. Captopril inhibited the vasoconstrictor responses to angiotensin I and [des-Asp1] angiotensin I, but did not affect the responses to angiotensins II or III, or norepinephrine. [Ile7] angiotensin III inhibited the vasoconstrictor responses to all four angiotensin peptides but did not alter the responses to norepinephrine. These findings indicate that the hindlimb vasoconstrictor responses to [des-Asp1] angiotensin I were due to the local formation of angiotensin III. The extent of conversion of [des-Asp1] angiotensin I to angiotensin III that occurred in one transit through the hindlimb arterial circulation was estimated to be 36.7%, which was not different from the estimated 36.4% conversion of angiotensin I to angiotensin II. We conclude that angiotensin I and [des-Asp1] angiotensin I are converted to their respective vasoactive forms (angiotensins II and III) to a similar extent in the hindlimb circulation via the action of kininase II.     

Involvement of angiotensin II and angiotensin IV in individual features of defensive and drinking behavior of rats

Involvement of angiotensin II and angiotensin IV in the defensive (tail flick test) and learned drinking behavior of rats was studied. It was found that angiotensin II was involved in drinking behavior of rats to a greater degree than angiotensin IV. On the contrary, angiotensin IV produced a stronger effect on the defensive behavior than angiotensin II. Individual features of rat behavior were revealed.     

Angiotensin converting enzyme 2 (ACE2) activity in fetal calf serum: implications for cell culture research

Cell culture experiments often employ the use of culture media that contain fetal calf serum (FCS). The angiotensin peptides angiotensin II and angiotensin 1–7 have opposing effects with angiotensin converting enzyme 2 (ACE2) being the enzyme predominantly responsible for generating angiotensin 1–7 from angiotensin II. The effect of FCS on angiotensin peptides has not previously been described. We have shown that FCS has ACE2 enzyme activity capable of degrading angiotensin II and generating angiotensin 1–7. Researchers should be aware that FCS possesses ACE2 activity and that heat-treating FCS to 56 °C only partially inhibits this enzyme activity, whereas heat-treating to 70 °C completely abolishes ACE2 activity.     

Effect of aldosterone on vascular angiotensin II receptors in the rat

The effect of aldosterone on the density and affinity of binding sites for 125I-labelled angiotensin II was investigated in a particulate fraction prepared from the rat mesenteric arteriolar arcades. The infusion of aldosterone 6.6 micrograms/h intraperitoneally via Alzet osmotic minipumps for 6 d produced an increase in the density of binding sites for 125I-labelled angiotensin II without change in affinity. After sodium depletion, mesenteric artery angiotensin II receptors were down-regulated as expected. An increase in the number of binding sites could be found when aldosterone was infused into sodium-depleted rats with no change in the elevated plasma renin activity. The intraperitoneal infusion of angiotensin II (200 ng X kg-1 X min-1 for 6 d) simultaneously with aldosterone resulted in down-regulation of vascular angiotensin II receptors, whereas after intravenous angiotensin II infusion (at 60 ng X kg-1 X min-1) the density of angiotensin II binding sites rose with aldosterone infusion. Plasma renin activity (PRA) was reduced and plasma angiotensin II increased in a dose-dependent fashion after angiotensin II infusion. An aldosterone concentration of 3 ng/mL for 18 h produced an increase in the number of angiotensin II binding sites in rat mesenteric artery smooth muscle cells in culture. We conclude that increased plasma aldosterone may result in up-regulation of vascular angiotensin II receptors independently of changes in plasma renin activity, and may in certain physiological states effectively antagonize the down-regulating action of angiotensin II.     

Angiotensin 'antipeptides': (-)messenger RNA complementary to human angiotensin II (+)messenger RNA encodes an angiotensin receptor antagonist

(-)mRNA complementary to human angiotensin II (+)mRNA encodes the 'antipeptide' Glu-Gly-Val-Tyr-Val-His-Pro-Val which is structurally related to angiotensin II. Angiotensin II 'antipeptide' (antiANG II) and the desglutamyl heptapeptide (antiANG III) are Type I antagonists which inhibit the contractile action of angiotensin at smooth muscle receptors by binding to a negative modulatory site on the angiotensin receptor which is distinct from the angiotensin binding site. These findings may illustrate that the inhibitory binding site on the angiotensin receptor exists to accomodate a naturally occurring inhibitor(s), which is encoded by the DNA strand complementary to that encoding angiotensin II.     

Measurement of immunoreactive angiotensin peptides in rat tissues: some pitfalls in angiotensin II analysis

Angiotensin II, the major effector peptide of the renin-angiotensin system, is an endocrine and paracrine regulator of tissue function. To determine its physiological role, it is important to quantify angiotensin II and related fragment peptides in tissues and plasma as a first step toward understanding angiotensin II metabolism within tissues. A fully characterized, sensitive, and reproducible immunochemical assay has been developed for quantitating angiotensin II immunoreactivity in tissues and plasma. We identified two methodological events of critical importance, incompletely addressed in previously reported studies. First, the nonspecific interference resulting from Sep-Pak processing was found to be due to hydrophobic impurities in the octade-casilane absorbent which were eliminated by washing the Sep-Pak with tetrahydrofuran and hexane before use. Second, a significant discrepancy was observed in the recoveries of angiotensin II and 125I-angiotensin II added to tissue extracts following high-pressure liquid chromatography. Angiotensin II immunoreactivity extracted from decapitated rat adrenal gland, brain, and kidney (target organs for angiotensin II), ovary and uterus (potential target organs for angiotensin II), and plasma has been characterized. The predominant component of the angiotensin II immunoreactivity was the biologically active octapeptide angiotensin II. However, in the brain, the ratio of angiotensin II to C-terminal angiotensin II immunoreactive fragments was lower than observed in other tissues studied. Other angiotensin II C-terminal immunoreactive peptide fragments-the biologically active heptapeptide and the biologically inactive angiotensin(3-8) and angiotensin(4-8)--were also detected in variable quantities in the various tissues.     

Low molecular weight angiotensin I converting enzyme from rat lung.

A low molecular weight angiotensin I converting enzyme (light angiotensin enzyme) was isolated from a homogenate of rat lung subjected to dialysis against sodium acetate at pH 4.8. This enzyme has a molecular weight of 84 000 on Sephadex G-200 and a molecular weight of 91 000 on SDS-poly-acrylamide gel as compared with a molecular weight of 139 000 for angiotensin I converting enzyme on SDS-polyacrylamide. Light angiotensin enzyme was activated by NaCl and inhibited by EDTA, angiotensin II, and bradykinin potentiating factor nonapeptide. Light angiotensin enzyme cross-reacted with antibody prepared against angiotensin I converting enzyme and stained with periodic acid-Schiff reagent as a glycoprotein. The evidence suggests that light angiotensin enzyme is a fragment of the higher molecular weight enzyme.     

Effects of angiotensin analogues and angiotensin receptor antagonists on paraventricular neurones.

In a previous study we observed that most neurones in the paraventricular nucleus are excited by angiotensin-(1-7). In comparison with angiotensin III this excitatory action was significantly delayed. The aim of the present microiontophoretic study of angiotensin II-sensitive rat paraventricular neurones was to compare the effect of the angiotensin-analogues angiotensin-(1-7), angiotensin-(2-7), angiotensin II and angiotensin III on the spontaneous activity of these neurones and to test angiotensin receptor subtype 1 antagonists (CGP 46027 or DuP 753) and subtype 2 selective antagonists (CGP 42112A and PD 123177) in order to acquire more evidence of the receptor subtype present. As previously observed angiotensin II, angiotensin III and angiotensin-(1-7) excited most neurones. The effect of angiotensin-(1-7) was usually weaker than that of angiotensin II, and in contrast to angiotensin III the latencies were not significantly different. Angiotensin-(1-7) seemed to be active by itself, because its effect was antagonised by angiotensin receptor antagonists. Angiotensin-(2-7) was mostly inactive, although a few cells were excited. Whereas the excitatory effects of angiotensin-(1-7), angiotensin II and angiotensin III could always be inhibited with both angiotensin receptor subtype antagonists 1 and 2, that produced by angiotensin-(2-7) was only weakly antagonised, if at all. Subtype 1 selective antagonists were effective at lower concentrations than selective subtype 2 antagonists.     

Rapamycin induces binding activity to the terminal oligopyrimidine tract of ribosomal protein mRNA in rats

Cathepsin G, elastase, and proteinase 3 are serine proteinases released by activated neutrophils. Cathepsin G can cleave angiotensinogen to release angiotensin II, but this activity has not been previously reported for elastase or proteinase 3. In this study we show that elastase and proteinase 3 can release angiotensin I from angiotensinogen and release angiotensin II from angiotensin I and angiotensinogen. The relative order of potency in releasing angiotensin II by the three proteinases at equivalent concentrations is cathepsin G > elastase > proteinase 3. When all three proteinases are used together, the release of angiotensin II is greater than the sum of the release when each proteinase is used individually. Cathepsin G and elastase can also degrade angiotensin II, reactions which might be important in regulating the activity of angiotensin II. The release and degradation of angiotensin II by the neutrophil proteinases are reactions which could play a role in the local inflammatory response and wound healing.     

Biosynthesis of Angiotensinogen and Angiotensins by Brain Cells in Primary Culture

This study focuses on the ability of primary rat brain cells in culture to synthesize angiotensinogen, angiotensin I, and angiotensin II. HPLC in combination with radioimmunoassay was used to characterize these compounds. Following incubation with 3H-labeled isoleucine, radioactively labeled angiotensinogen with an approximate molecular weight of 25,000 was identified in both glial and neuronal cells. Other molecular weight forms of angiotensinogen with molecular weights of about 300 and 160,000 were present in both cell types. In addition to angiotensinogen, radioactively labeled angiotensin I and angiotensin II were also synthesized by neuronal and glial cells. These results suggest that glial and neuronal cells can synthesize angiotensinogen, angiotensin I, and angiotensin II in a similar manner shown for the peripheral renin angiotensin system.     

Angiotensin receptors: form and function and distribution

The peptide hormone, angiotensin II, acts primarily via type I (AT(1)) and type II (AT(2)) angiotensin receptors. Proteolytic fragments of angiotensin II also have biological activity via these and other receptors, with actions that may mimic or antagonise angiotensin II. Most notably, a high affinity-binding site for angiotensin IV (the Val(3)-Phe(8) fragment of angiotensin II) has recently been identified as the insulin-regulated aminopeptidase (IRAP). While AT(1) and AT(2) receptors are seven transmembrane-spanning, G protein-coupled receptors with some well-established features of relevance to health and disease, the existence of separate receptor systems for angiotensin fragments offers exciting possibilities for new therapeutics to target the diverse actions of the angiotensin peptides.     

Angiotensin receptor type 1 forms a complex with the transient outward potassium channel Kv4.3 and regulates its gating properties and intracellular localization

We report a novel signal transduction complex of the angiotensin receptor type 1. In this complex the angiotensin receptor type 1 associates with the potassium channel alpha-subunit Kv4.3 and regulates its intracellular distribution and gating properties. Co-localization of Kv4.3 with angiotensin receptor type 1 and fluorescent resonance energy transfer between those two proteins labeled with cyan and yellow-green variants of green fluorescent protein revealed that Kv4.3 and angiotensin receptor type I are located in close proximity to each other in the cell. The angiotensin receptor type 1 also co-immunoprecipitates with Kv4.3 from canine ventricle or when co-expressed with Kv4.3 and its beta-subunit KChIP2 in human embryonic kidney 293 cells. Treatment of the cells with angiotensin II results in the internalization of Kv4.3 in a complex with the angiotensin receptor type 1. When stimulated with angiotensin II, angiotensin receptors type 1 modulate gating properties of the remaining Kv4.3 channels on the cell surface by shifting their activation voltage threshold to more positive values. We hypothesize that the angiotensin receptor type 1 provides its internalization molecular scaffold to Kv4.3 and in this way regulates the cell surface representation of the ion channel.     

Effects of platelet-activating factor on conversion of angiotensin I to II

We have studied the effects of platelet-activating factor (alkyl-acetyl-GPC) on conversion of angiotensin I to II. Platelet-activating factor (PAF) had a novel effect on angiotensin I conversion. Pulmonary artery endothelial cells converted 30% of [125I]angiotensin I to angiotensin II in the absence of PAF, but their activity was greatly stimulated in its presence. When PAF was added to pulmonary artery endothelial cells, the conversion of angiotensin I to II was enhanced up to 68%. Maximal stimulation without cellular damage was achieved at 10(-5) M PAF. LysoPAF did not stimulate the conversion of angiotensin I to angiotensin II at any concentration used.     

Stimulation of platelet-derived growth factor-induced DNA synthesis by angiotensin II in rabbit vascular smooth muscle cells

In cultured rabbit vascular smooth muscle cells (VSMCs), angiotensin II by itself had little mitogenic effect even in the presence of cell-free plasma-derived serum (PDS), but markedly stimulated the platelet-derived growth factor (PDGF)-induced DNA synthesis in the presence of PDS. The maximal extent of DNA synthesis induced by PDGF plus angiotensin II was about twice that induced by PDGF alone. The stimulatory effect of angiotensin II was dose-dependent with the maximal response seen at 1 microM and was inhibited by the specific angiotensin II receptor antagonist, [Sar1, Ile8]angiotensin II. In VSMCs, both PDGF and angiotensin II induced expression of the c-fos gene in dose-dependent manners. In contrast to the synergistic effect of angiotensin II and PDGF on DNA synthesis, they induced expression of the c-fos gene in an additive manner. These results suggest that angiotensin II may act as a growth regulator for VSMCs in addition to acting as a vasoconstrictor.     

Effects of angiotensin I and II and their interactions with some prostanoids in perfused human umbilical arteries

In perfused human umbilical arteries both angiotensin I and II induced vasoconstriction with a monophasic response. Angiotensin I and II induced vasoconstrictions at doses greater than or equal to 10(-8) M and 10(-9) M respectively. Captopril inhibited the angiotensin I response while the angiotensin II receptor blocker Sar1-Ala8 AII inhibited the effect of both angiotensins. PGI2 attenuated the angiotensin II response in a dose dependent pattern. PGE2 and PGF2 alpha in concentrations below the critical levels for creating pressure responses per se, also attenuated the angiotensin II response. The cyclooxygenase inhibitor indomethacin potentiated the angiotensin II response indicating that endogenous production of prostanoids is of importance in the modulation of angiotensin effects.     

Metabolism of angiotensin I in the coronary circulation of normal and diabetic rats

Formation of metabolites from angiotensin I that passed the coronary vessels in the isolated working rat hearts of normal and streptozotocin-induced diabetes was evaluated. HPLC analysis showed that the levels of angiotensin II and angiotensin 1-7 were unaltered in the diabetic hearts, but the perfusates of the diabetic hearts contained smaller amounts of angiotensin 1-9. It is not clear why the perfusates of diabetic hearts contain less amount of angiotensin 1-9. It is possible that the peptide is metabolized faster or greater internalization takes place in the diabetic heart. The amount of angiotensin II in the perfusates of normal hearts was 5.8 times greater at the perfusion rate of 2 than at 10 ml/min/g wet heart weight. At such conditions, the amount of angiotensin 1-9 and angiotensin 1-7 in the perfusates were increased 2.4 and 1.5 times, respectively. A higher amount of angiotensin II during myocardial hypoperfusion may lead to constriction of the coronary vessels. As a result, myocardial damage may be facilitated.     

Changes of Blood Pressure,Renin, and Angiotensin after Bilateral Nephrectomy in Patients with Chronic Renal Failure

Circulating levels of renin, angiotensin I, and angiotensin II were increased in six patients with chronic renal failure and hypertension uncontrolled by dialysis and hypotensive drugs. Lower and often normal levels were found in 10 patients whose blood pressure was controlled by dialysis treatment. For a variety of reasons all patients were subjected to bilateral nephrectomy. The logarithm of the decrease in plasma concentrations of renin and angiotensin II was significantly related to the fall of blood pressure after operation. Plasma renin concentration correlated significantly with blood angiotensin I concentration and with plasma angiotensin II in samples taken before and after nephrectomy. Renin, angiotensin I, and angiotensin II were measurable in samples of blood taken 48 hours or more after the operation.     

Angiotensin II binding to mammalian brain membranes.

High affinity binding sites for angiotensin II in bovine and rat brain membranes have been identified and characterized using monoiodinated Ile5-angiotensin II of high specific radioactivity. Degradation of labeled and unlabeled peptide by washed brain particulate fractions was prevented by adding glucagon to the final incubation medium and including a proteolytic enzyme inhibitor (phenylmethylsulfonyl fluoride) in preincubation and incubation procedures. 125I-Angiotensin II binding can be studied using either centrifugation or filtration techniques to separate tissue-bound radioactivity. 125I-Angiotensin II binding to calf brain membranes is saturable and reversible, with a dissociation binding constant of 0.2 nM at 37 degrees. A similar binding constant is found in rat brain membranes. Analogues and fragments of angiotensin II compete for these brain binding sites with potencies which correlate with both their in vivo potencies and their binding inhibition protencies at adrenal cortex angiotensin II receptors. Angiotensin I is 1 to 2 orders of magnitude weaker than angiotensin II; the 3-8 hexapeptide and 4-8 pentapeptide are much weaker still. (desAsp1) angiotensin II (angiotensin III) is slightly more potent than angiotensin II, as are several antagonists of angiotensin II with aliphatic amino acids substituted at position 8. In calf brain 125I-angiotensin II binding is restricted almost exclusively to the cerebellum (cortex and deep nuclei). In rat brain, angiotensin II binding is highest in the thalamus-hypothalamus, midbrain, and brainstem, areas which are believed to be involved in mediating angiotensin II-induced central effects. These findings illustrate the presence of high affinity specific binding sites for angiotensin II in rat and bovine brain and suggest a physiological role for angiotensin peptides in the central nervous system.