Angiotensins II and IV stimulate the activity of tyrosine kinases in estrogen-induced rat pituitary tumors

The effects of angiotensin II (AngII) and its fragment 3-8 (angiotensin IV, AngIV) on tyrosine kinase (TK) activity in estrogen-induced rat pituitary tumor homogenates were studied. It was found that both angiotensin peptides increase the TK activity. AngIV was effective in a lower concentration and its maximal effect was greater as compared to that of AngII. Moreover, the specific inhibitors of aminopeptidase A (EC33) and of aminopeptidase N (PC18) significantly attenuated the stimulatory effect of AngII. Because the action of both aminopeptidases is necessary to convert AngII into AngIV, this finding suggested that the effect of AngII on TK activity is (at least in part) exerted via AngIV.     

Angiotensin II protects against alpha-synuclein toxicity and reduces protein aggregation in vitro

In this study, we examined the effects of angiotensin II (AngII) in a genetic in vitro PD model produced by alpha-synuclein (alpha-syn) overexpression in the human neuroglioma H4 cell line. We observed a maximal decrease in alpha-syn-induced toxicity of 85% and reduction in inclusion formation by 19% when cultures were treated with AngII in the presence of the angiotensin type 1 (AT1) receptor antagonist losartan and AT2 receptor antagonist PD123319. When compared to AngII, the AT4 receptor agonist AngIV was moderately effective in protecting H4 cells against alpha-syn toxicity and did not significantly reduce inclusion formation. Here we show that AngII is protective against genetic, as well as neurotoxic models of PD. These data support the view that agents acting on the renin-angiotensin-system (RAS) may be useful in the prevention and/or treatment of Parkinson's disease.     

Modulation of the renin-angiotensin system may alter the adrenocortical regeneration

The effect of the renin-angiotensin system on adrenocortical regeneration has been studied in rats subjected to left adrenal enucleation combined with contralateral adrenalectomy. It was found that angiotensin II stimulated both proliferation and the steroidogenic capacity of the regenerating adrenal cortex cells by 6 days after operation. The stimulatory effect of angiotensin was prevented by losartan, a type 1 angiotensin (AT1) receptor antagonist, and by nifedipine, a calcium channel blocker. Losartan and nifedipine also depressed the adrenocortical regeneration when given alone. Enalapril, an angiotensin converting enzyme blocker, inhibited both the proliferation and steroidogenesis of the regenerating adrenal cortex, but this effect could be prevented by angiotensin II.     

A role for the angiotensin IV/AT4 system in mediating natriuresis in the rat

Angiotensin II (AngII) or Angiotensin IV (AngIV) was infused into the renal artery of anesthetized rats while renal cortical blood flow was measured via laser Doppler flowmetry. The infusion of AngII produced a significant elevation in mean arterial pressure (MAP) with an accompanying decrease in cortical blood flow, glomerular filtration rate (GFR), urine volume, and urine sodium excretion. The infusion of AngIV induced significant increases in renal cortical blood flow and urine sodium excretion, without altering MAP, GFR, and urine volume. Pretreatment infusion with a specific AT1 receptor antagonist, DuP 753, blocked or attenuated the subsequent AngII effects, while pretreatment infusion with the specific AT4 receptor antagonist, Divalinal-AngIV, blocked the AngIV effects. These results support distinct and opposite roles for AngII and AngIV, i.e. AngII acts as an anti-natriuretic agent, while AngIV acts as a natriuretic agent.     

The involvement of angiotensins in the control of prostatic epithelial cell proliferation in the rat.

The effects of captopril (the inhibitor of the angiotensin-converting enzyme) and of angiotensins II and IV (3-8 fragment of angiotensin II) on cell proliferation of the prostatic epithelium was investigated in the rat. The incorporation of bromodeoxyuridine into cell nuclei was used as an index of cell proliferation. It was found that the treatment with captopril resulted in the suppression of prostatic epithelial cell proliferation. The antiproliferative effect of captopril was reversed (at least partially) by a simultaneous treatment with either angiotensin II or angiotensin IV. The effects of angiotensins were not blocked by the administration of losartan--AT1 angiotensin receptor blocker. These findings suggest the involvement of angiotensins in the control of prostatic growth, acting via the receptors different from the AT1-subtype (presumably via AT4 receptors).     

Intracrine action of angiotensin II in mesangial cells: subcellular distribution of angiotensin II receptor subtypes AT1 and AT2

Biological effects of angiotensin II (AngII) such as regulation of AngII target genes may be triggered by interaction of AngII with intracellular AngII receptor types 1 and 2 (AT₁ and AT₂), defined as intracrine response. The aim of this study was to examine the presence of AT₁ and AT₂ receptors in nuclear membrane of human mesangial cells (HMCs) and evaluate the possible biological effects mediated by intracellular AT₁ through an intracrine mechanism. Subcellular distribution of AT₁ and AT₂ was evaluated by immunofluorescence and by western blot in isolated nuclear extract. Endogenous intracellular synthesis of AngII was stimulated by high glucose (HG). Effects of HG were analyzed in the presence of candesartan, which prevents AngII internalization. Both receptors were found in nuclear membrane. Fluorescein isothiocyanate (FITC)-labeled AngII added to isolated nuclei produced a fluorescence that was reduced in the presence of losartan or PD-123319 and quenched in the presence of both inhibitors simultaneously. HG induced overexpression of fibronectin and increased cell proliferation in the presence of candesartan, indicating an intracrine action of AngII induced by HG. Results showed the presence of nuclear receptors in HMCs that can be activated by AngII through an intracrine response independent of cytoplasmic membrane AngII receptors.

     

Cardiac fibrogenesis in magnesium deficiency: a role for circulating angiotensin II and aldosterone

Mechanisms underlying cardiac fibrogenesis in magnesium deficiency are unclear. It was reported earlier from this laboratory that serum from magnesium-deficient rats has a more pronounced stimulatory effect on cell proliferation, net collagen production, and superoxide generation in adult rat cardiac fibroblasts than serum from rats on the control diet. The profibrotic serum factors were, however, not identified. This study tested the hypothesis that circulating angiotensin II may modulate cardiac fibroblast activity in hypomagnesemic rats. Male Sprague-Dawley rats were pair-fed a magnesium-deficient (0.0008% Mg) or -sufficient (0.05%) diet for 6 days, and the effects of serum from these rats on [3H]thymidine and [3H]proline incorporation into cardiac fibroblasts from young adult rats were evaluated in the presence of losartan, an angiotensin II type 1 (AT1) receptor antagonist, and spironolactone, an aldosterone antagonist. Losartan and spironolactone markedly attenuated the stimulatory effects in vitro of serum from the magnesium-deficient and control groups, but the inhibitory effects were considerably higher in cells exposed to serum from magnesium-deficient animals. Circulating and cardiac tissue levels of angiotensin II were significantly elevated in magnesium-deficient animals (67.6% and 93.1%, respectively, vs. control). Plasma renin activity was 61.9% higher in magnesium-deficient rats, but serum angiotensin-converting enzyme activity was comparable in the two groups. Furthermore, preliminary experiments in vivo using enalapril supported a role for angiotensin II in magnesium deficiency. There was no significant difference between the groups in serum aldosterone levels. The findings suggest that circulating angiotensin II and aldosterone may stimulate fibroblast activity and contribute to a fibrogenic response in the heart in magnesium deficiency.     

A Specific Binding Site for Angiotensin II(3-8), Angiotensin IV,in Rabbit Cardiac Fibroblasts

Abstract

This study demonstrates the existence of a high affinity binding site on rabbit cardiac fibroblasts of the hexapeptide (3-8) fragment of angiotensin II (AngIV). [¹²⁵I]-AngIV binding is saturable, reversible and distinct from angiotensin II (AngII) receptors. At 37°C equilibrium of [¹²⁵I]-AngIV binding is reached within 2 h. AngIV displaces [¹²⁵I]-AngIV bound to cultured rabbit cardiac fibroblasts whereas AngII receptor-specific ligands ([Sar¹,IIe⁸]-AngII, Dup753, CGP42112A) do not. Scatchard plot analysis revealed that [¹²⁵I]-AngIV binds to a single class of sites with K_d = 4.87 ± 0.11 × 10^?9 mol/l, B_max = 371 ± 8.3 fmol/mg protein and a Hill coefficient of 0.92. In the presence of the non-hydrolyzable GTP analog GTP_γS [¹²⁵I]-AngIV binding in rabbit cardiac fibroblasts was not markedly affected, whereas binding of [¹²⁵I]-(Sar¹,IIe⁸)-AngII is reduced. The role of AngIV in the heart and in particular in cardiac fibroblasts is unknown, and the putative interaction of AngIV with AngII needs further characterization.     

The role of AT1 receptor-mediated reproductive function in renovascular hypertension in male rats

There is an association between hypertension and reproductive dysfunction. Angiotensin II (Ang II) is involved in the pathogenesis of hypertension and the regulation of reproduction. The present study aimed to determine whether the angiotensinergic system mediates the effects of hypertension on reproductive function in male rats subjected to a two-kidney, one-clip (2K1C) model. Sexual behavior parameters, gametogenesis and plasma concentrations of Ang II, testosterone, prolactin and corticosterone were evaluated in male rats 28days after 2K1C or sham surgery and losartan (Los) treatment (a type 1 angiotensin II (AT1) receptor antagonist) or vehicle (V) treatment. The animals were divided into Sham+V, 2K1C+V, Sham+Los and 2K1C+Los groups. The 2K1C+V group showed a hypertensive response, inhibition of sexual behavior, spermatogenesis dysfunction, and increases in plasma Ang II and prolactin. Conversely, plasma testosterone decreased, and plasma corticosterone remained constant. Losartan treatment normalized blood pressure and prevented the changes in plasma testosterone and prolactin, sexual behavior and spermatogenesis in the 2K1C+Los group. In addition, losartan treatment caused an additional increase in circulating Ang ll in both groups (Sham+Los and 2K1C+Los). Together, these results suggest that Ang ll, acting through the AT1 receptor, modulates behavioral and endocrine parameters of reproductive function during renovascular hypertension. In addition, the effects of circulating Ang II on plasma testosterone and prolactin seem to contribute to the spermatogenic and sexual dysfunctions in hypertensive rats.     

Oxidative stress in the infarcted heart: role of de novo angiotensin II production

This study aimed to elucidate whether angiotensin (Ang) II generated de novo at the infarct site regulates the redox state of inflammatory cells participating in cardiac repair. On days 3-28 following ligation of the rat left coronary artery, we addressed at the infarct site: (a) the appearance and cellular origin of oxidative stress by monitoring the expression (mRNA and protein) of gp91phox (a subunit of superoxide producing NADPH oxidase) and the antioxidant superoxide dismutase (SOD), together with the presence of 3-nitrotyrosine (a marker of oxidative stress); (b) the presence of components requisite to Ang peptide generation; and (c) response to treatment with losartan (Los, 10mg/kg/day). We found at the infarct site, macrophage-derived oxidative stress appears during week 1 coincident with the appearance of components requisite to AngII generation and activity in these cells. Based on observed response to AT1 receptor antagonism with Los, we would suggest de novo AngII, in an autocrine manner, participates in the induction of oxidative stress while suppressing the expression of antioxidant defenses.     

Angiotensin II Reduces Cardiac AdipoR1 Expression through AT1 Receptor/ROS/ERK1/2/c-Myc Pathway

Adiponectin, an abundant adipose tissue-derived protein, exerts protective effect against cardiovascular disease. Adiponectin receptors (AdipoR1 and AdipoR2) mediate the beneficial effects of adiponectin on the cardiovascular system. However, the alteration of AdipoRs in cardiac remodeling is not fully elucidated. Here, we investigated the effect of angiotensin II (AngII) on cardiac AdipoRs expression and explored the possible molecular mechanism. AngII infusion into rats induced cardiac hypertrophy, reduced AdipoR1 but not AdipoR2 expression, and attenuated the phosphorylations of adenosine monophosphate-activated protein kinase and acetyl coenzyme A carboxylase, and those effects were all reversed by losartan, an AngII type 1 (AT1) receptor blocker. AngII reduced expression of AdipoR1 mRNA and protein in cultured neonatal rat cardiomyocytes, which was abolished by losartan, but not by PD123319, an AT2 receptor antagonist. The antioxidants including reactive oxygen species (ROS) scavenger NAC, NADPH oxidase inhibitor apocynin, Nox2 inhibitor peptide gp91 ds-tat, and mitochondrial electron transport chain complex I inhibitor rotenone attenuated AngII-induced production of ROS and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. AngII-reduced AdipoR1 expression was reversed by pretreatment with NAC, apocynin, gp91 ds-tat, rotenone, and an ERK1/2 inhibitor PD98059. Chromatin immunoprecipitation assay demonstrated that AngII provoked the recruitment of c-Myc onto the promoter region of AdipoR1, which was attenuated by PD98059. Moreover, AngII-induced DNA binding activity of c-Myc was inhibited by losartan, NAC, apocynin, gp91 ds-tat, rotenone, and PD98059. c-Myc small interfering RNA abolished the inhibitory effect of AngII on AdipoR1 expression. Our results suggest that AngII inhibits cardiac AdipoR1 expression in vivo and in vitro and AT1 receptor/ROS/ERK1/2/c-Myc pathway is required for the downregulation of AdipoR1 induced by AngII.     

Comparative effects of angiotensin IV and two hemorphins on angiotensin-converting enzyme activity

The role of angiotensin IV (AngIV) in the regulation of angiotensin-converting enzyme (ACE) was studied in vitro. This study demonstrates that this active fragment appeared as a novel endogenous ACE inhibitor. Inhibitory kinetic studies revealed that AngIV acts as a purely competitive inhibitor with a K(i) value of 35 microM. AngIV was found to be quite resistant to ACE hydrolysis opposite to hemorphins which are both ACE inhibitors and substrates. In order to confirm a putative role of AngIV and hemorphins in the Renin-Angiotensin system (RAS) regulation, we studied their influence on AngI conversion. We noticed that 16.7 microM of both peptides decreased more than 50% of AngI conversion to AngII in vitro. The capacity of hemorphins, particularly LVVH-7, and AngIV to inhibit ACE activity here suggests a synergistic relation between these two peptides and the regulation of RAS.     

Angiotensin II inhibits bTREK-1 K+ channels in adrenocortical cells by separate Ca2+- and ATP hydrolysis-dependent mechanisms

Bovine adrenocortical cells express bTREK-1 K+ channels that set the resting membrane potential (V(m)) and couple angiotensin II (AngII) and adrenocorticotropic hormone (ACTH) receptors to membrane depolarization and corticosteroid secretion. In this study, it was discovered that AngII inhibits bTREK-1 by separate Ca2+- and ATP hydrolysis-dependent signaling pathways. When whole cell patch clamp recordings were made with pipette solutions that support activation of both Ca2+- and ATP-dependent pathways, AngII was significantly more potent and effective at inhibiting bTREK-1 and depolarizing adrenal zona fasciculata cells, than when either pathway is activated separately. External ATP also inhibited bTREK-1 through these two pathways, but ACTH displayed no Ca2+-dependent inhibition. AngII-mediated inhibition of bTREK-1 through the novel Ca2+-dependent pathway was blocked by the AT1 receptor antagonist losartan, or by including guanosine-5'-O-(2-thiodiphosphate) in the pipette solution. The Ca2+-dependent inhibition of bTREK-1 by AngII was blunted in the absence of external Ca2+ or by including the phospholipase C antagonist U73122, the inositol 1,4,5-trisphosphate receptor antagonist 2-amino-ethoxydiphenyl borate, or a calmodulin inhibitory peptide in the pipette solution. The activity of unitary bTREK-1 channels in inside-out patches from adrenal zona fasciculata cells was inhibited by application of Ca2+ (5 or 10 microM) to the cytoplasmic membrane surface. The Ca2+ ionophore ionomycin also inhibited bTREK-1 currents through channels expressed in CHO-K1 cells. These results demonstrate that AngII and selected paracrine factors that act through phospholipase C inhibit bTREK-1 in adrenocortical cells through simultaneous activation of separate Ca2+- and ATP hydrolysis-dependent signaling pathways, providing for efficient membrane depolarization. The novel Ca2+-dependent pathway is distinctive in its lack of ATP dependence, and is clearly different from the calmodulin kinase-dependent mechanism by which AngII modulates T-type Ca2+ channels in these cells.     

Developmental changes in cell proliferation in the auditory midbrain of the bullfrog, Rana catesbeiana

We examined patterns of cell proliferation in the auditory midbrain (torus semicircularis) of the bullfrog, Rana catesbeiana, over larval and early postmetamorphic development, by visualizing incorporation of 5-bromo-2'-deoxyuridine (BrdU) in cycling cells. At all developmental stages, BrdU-labeled cells were concentrated around the optic ventricle. BrdU-labeled cells also appeared within the torus semicircularis itself, in a stage-specific manner. The mitotic index, quantified as the percent of BrdU-positive cells outside the ventricular zone per total cells available for label, varied over larval development. Mitotic index was low in hatchling, early larval, and late larval stages, and increased significantly in deaf period, metamorphic climax, and froglet stages. Cell proliferation was higher in metamorphic climax than at other stages, suggesting increased cell proliferation in preparation for the transition from an aquatic to an amphibious existence. The change in mitotic index over development did not parallel the change in the total numbers of cells available for label. BrdU incorporation was additionally quantified by dot-blot assay, showing that BrdU is available for label up to 72 h postinjection. The pattern of change in cell proliferation in the torus semicircularis differs from that in the auditory medulla (dorsal medullary nucleus and superior olivary nucleus), suggesting that cell proliferation in these distinct auditory nuclei is mediated by different underlying mechanisms.     

Flow cytometric determination of cell proliferation in hypertensive blood vessels.

BACKGROUND: Measurement of vascular cell proliferation in animal models of hypertension is currently accomplished by demonstrating [(3)H]-thymidine ([(3)H]-dT) incorporation into DNA using autoradiography. This method, however, is labor intensive, requires radioactivity, and is limited by the inherent difficulty in discriminating labeled and unlabeled cells. To address these limitations, a flow cytometric-based method is described utilizing incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA of nuclei isolated from blood vessels. METHODS: Pulmonary hypertension was induced in rats by exposure to 10% O(2) (hypoxia) for varying periods of time. Pulmonary arteries and aorta from rats injected with BrdU prior to sacrifice were isolated, fixed with 10% formalin, and digested with Protease XIV. The intact nuclei liberated by this treatment were successively treated with HCl/Triton X-100 and sodium borate. Processed nuclei were probed with a BrdU-specific fluorescein-conjugated antibody, and the percentage of BrdU staining cells was determined using flow cytometry. RESULTS: An approximately 20-fold increase in BrdU-positive cells at 3 days of hypoxia in pulmonary arteries (relative to control) with no change in aorta was observed. These results were similar to previous studies using [(3)H]-dT labeling. CONCLUSIONS: Flow cytometric determination of cell proliferation in blood vessels is a simple, objective technique that may facilitate measurement of cell proliferation in animal models of vascular disease.     

Synthetic modified N-POMC1–28 controls in vivo proliferation and blocks apoptosis in rat adrenal cortex

The identity of the pro-opiomelanocortin (POMC)-derived mitogen in the adrenal cortex has been historically controversial. We have used well-established in vivo models, viz., hypophysectomized (Hyp) or dexamethasone (Dex)-treated rats, to study the effect of the synthetic modified peptide N-terminal POMC (N-POMC_1–28) on DNA synthesis in the adrenal cortex, as assessed by BrdU incorporation and compared with adrenocorticotropic hormone (ACTH). We evaluated the importance of disulfide bridges on proliferation by employing N-POMC_1–28 without disulfide bridges and with methionines replacing cysteines. Acute administration of synthetic modified N-POMC_1–28 distinctly increased DNA synthesis in the zona glomerulosa and zona fasciculata, but not in the zona reticularis in Hyp rats, whereas in Dex-treated rats, this peptide was effective in all adrenal zones. ACTH administration led to an increase of BrdU-positive cells in all adrenal zones irrespective of the depletion of Hyp or Dex-POMC peptides. The use of the ACTH antagonist, ACTH_7–38, confirmed the direct participation of ACTH in proliferation. Two different approaches to measure apoptosis revealed that both peptides similarly exerted a protective effect on all adrenocortical zones, blocking the apoptotic cell death induced by hypophysectomy. Thus, ACTH_1–39 and N-POMC_1–28 have similar actions suggesting that the disulfide bridges are important but not essential. Both peptides seem to be important factors determining adrenocortical cell survival throughout the adrenal cortex, reinforcing the idea that each zone can be renewed from within itself.     

Brain renin angiotensin system (RAS) in stress-induced analgesia and impaired retention.

Physiological stress is known to produce analgesia and memory disruption. Brain renin angiotensin system (RAS) has been reported to participate in stress response and plays a role in the processing of sensory information. Angiotensin receptors (AT), particularly AT1 subtypes have been reported to be distributed in brain areas that are intimately associated with stress response. The purpose of present study was to examine the modulation of AT1 receptor in the immobilization stress and angiotensin II (AngII)-induced analgesia and impaired retention, and to determine whether resultant behavioral changes involve common sensory signals. Result of present experiments showed that immobilization stress in mice and rats, and intracerebroventricular (ICV) administration of AngII (10 and 20 ng) in rats produced an increase in tail-flick latency. Similarly, post training administration of AngII or immobilization stress produced impairment of retention tested on plus-maze learning and on passive avoidance step-down task. Both these responses were sensitive to reversal by prior treatment with losartan (10 and 20 mg/kg), an AT1 AngII receptor antagonist. On the other hand, naloxone, an opiate antagonist preferentially attenuated the stress and AngII-induced analgesia and retention deficit induced by immobilization stress, but failed to reverse the AngII induced retention deficit. These results suggest immobilization stress-induced analgesia and impaired retention involves the participation of brain RAS. Further, failure of naloxone to reverse AngII-induced retention impairment shows. AngII-induced behavioral changes are under control of different sensory inputs.     

Effects of des-aspartate-angiotensin I on angiotensin II-induced incorporation of phenylalanine and thymidine in cultured rat cardiomyocytes and aortic smooth muscle cells

Des-aspartate-angiotensin I, a pharmacologically active nine-amino acid angiotensin peptide, and losartan, an AT(1) angiotensin receptor antagonist, but not angiotensin-(1-7), another active angiotensin peptide, completely attenuated the angiotensin II-induced incorporation of [3H]phenylalanine in cultured rat cardiomyocytes. The attenuation by des-aspartate-angiotensin I but not that of losartan was inhibited by indomethacin. The data support an earlier suggestion that the nonapeptide attenuates cardiac hypertrophy in rats via an indomethacin-sensitive angiotensin AT(1) receptor subtype. In rat aortic smooth muscle cells, both des-aspartate-angiotensin I and angiotensin-(1-7) had no effect on the angiotensin II-induced [3H]phenylalanine incorporation. However, the two peptides significantly attenuated the angiotensin II-induced [3H]thymidine incorporation in the smooth muscle cells. The attenuation by angiotensin-(1-7) but not by des-aspartate-angiotensin I was inhibited by (D-Ala(7))-angiotensin-(1-7), a specific angiotensin-(1-7) antagonist. Des-aspartate-angiotensin I also attenuated FCS-stimulated [3H]thymidine incorporation. This attenuation was inhibited by the peptide angiotensin receptor antagonist, (Sar(1), Ile(8))-angiotensin II, but not by losartan. These data indicate that des-aspartate-angiotensin I and angiotensin-(1-7) do not participate in the process of protein synthesis in vascular smooth muscle cells and that the nonapeptide and heptapeptide act on different non-AT(1) receptors to mediate their anti-hyperplasic action. Although the exact mechanisms of action remain to be elucidated, the findings indicate that des-aspartate-angiotensin I acts as an agonist on angiotensin AT(1) and non-AT(1) receptor subtypes and induces responses that oppose the actions of angiotensin II.