Angiotensin II regulates tyrosine hydroxylase activity and mRNA expression in rat mediobasal hypothalamic cultures: the role of specific protein kinases

Dopamine secreted by hypothalamic neurons is crucial in regulating prolactin secretion from the pituitary. We have examined the ability of angiotensin II (AngII) to regulate the activity of these dopaminergic neurons and thus act as a potential physiological regulator of prolactin secretion. Using a hypothalamic cell culture preparation we determined the effect of AngII on tyrosine hydroxylase activity and expression (TOH). This is important because TOH is the rate-limiting enzyme in dopamine biosynthesis. AngII stimulated a time- and concentration-dependent increase in TOH activity which was suppressed by inhibitors able to act on protein kinase A (PKA), protein kinase C (PKC) and Ca(2+)/calmodulin-dependent protein kinase II (CaMPKII). An inhibitor of the mitogen-activated protein kinase (MAPK) pathway, PD 98059, reduced basal TOH activity but the AngII response was still detectable. AngII stimulation enhanced the phosphorylation of TOH at Ser19, Ser31 and Ser40. AngII also induced a time-dependent increase in TOH mRNA expression which was unaffected by inhibitors able to act on PKA and CaMPKII, but was abolished by inhibitors able to act on ERK and PKC. AngII responses were very much larger in cultures prepared from female when compared to male rat pups. Data from adult hypothalamic slices confirmed this sexual dimorphism and supported the role of the protein kinases noted above. Therefore AngII can regulate both the activity and expression of TOH in hypothalamic neurons employing multiple, but only partially overlapping, signaling pathways.     

NMDA Receptors in the Medial Zona Incerta Stimulate Luteinizing Hormone and Prolactin Release

1. The aim of the present work is to demonstrate the interaction between the glutamatergic/NMDA and dopaminergic systems in the medial zona incerta on the control of luteinizing hormone and prolactin secretion and the influence of reproductive hormones. 2. Proestrus and ovariectomized rats were primed with estrogen and progesterone to induce high or low levels of luteinizing hormone and prolactin. 2-Amino-7-phosphonoheptanoic acid, an NMDA receptor antagonist, and dopamine were injected in the medial zona incerta. Blood samples were withdrawn every hour between 1,600 and 2,000 hours or 2,200 hours via intracardiac catheter from conscious rats. Additional groups of animals injected with the NMDA receptor antagonist were killed 1 or 4 h after injection. Dopamine and its metabolite 3,4-dihydroxyphenylacetic acid were measured in different hypothalamic regions. 3. 2-Amino-7-phosphonoheptanoic acid blocked the ovulatory luteinizing hormone surge in proestrus rats. 2-Amino-7-phosphonoheptanoic acid also blocked the increase in luteinizing hormone induced by ovarian hormones in ovariectomized rats, an effect that was partially reversed by dopamine injection. Conversely, the increased release of luteinizing hormone and prolactin induced by dopamine was prevented by 2-amino-7-phosphonoheptanoic acid. We found that the NMDA antagonist injection decreased the dopaminergic activity--as evaluated by the 3,4-dihydroxyphenylacetic acid/dopamine ratio--in the medio basal hypothalamus and increased in the preoptic area. 4. Our results show an stimulatory role of NMDA receptors on the ovulatory luteinizing hormone release and on luteinizing hormone release induced by sexual hormones and demonstrate that the stimulatory effect of dopamine on luteinizing hormone and prolactin is mediated by the NMDA receptors. These results suggest a close interaction between the glutamatergic and dopaminergic incertohypothalamic systems on the control of luteinizing hormone and prolactin release.     

Angiotensin II mediates Tyr-dephosphorylation in rat fetal kidney membranes

Angiotensin II (Ang II) elicits a variety of physiological effects through specific Ang II receptors in numerous tissues. In addition, Ang II is a modulator of cellular growth and exerts a positive or negative effect on cell growth depending on which receptor subtype is activated. Expression of the intrarenal AT₂ receptors occurs at its highest levels in the fetal kidney, with a rapid decline after birth. In the present paper, we performed a study on the signaling mechanism of Ang II receptors in rat fetal (E20) kidney, a rich source of AT₂ receptors, where both Ang II receptor subtypes are present. Ang II induces Tyr-dephosphorylation of proteins in rat fetal kidney membranes. The response is dose-dependent, with a reduction of 20% with respect to the control (100%), signal that is completely reversed by Ang II AT₂ competitor PD123319. Orthovanadate, the inhibitor of phospho-Tyr-phosphatases (PTPase), reverts Ang II effect, suggesting the involvement of a protein tyrosine phosphatase. The peptide analog of Ang II, CGP42112, exhibits an agonist effect, which is dose-dependent. Thus, in rat fetal (E20) kidney, the Ang-induced protein Tyr-dephosphorylation of several proteins is mediated by AT₂ receptors, mechanism that involves an orthovanadate sensitive PTPase.     

Angiotensin II suppresses adriamycin-induced apoptosis through activation of phosphatidylinositol 3-kinase/Akt signaling in human breast cancer cells

Angiotensin II (Ang II) stimulates tumor growth and angio-genesis in some solid cancer cells, but its anti-apoptosis role in breast cancer remains unclear. To address this issue, we investigated the effect of Ang II on adriamycin-induced apoptosis in breast cancer MCF-7 cells. Treatment of human breast cancer MCF-7 cells with adriamycin, a DNA topoisomerase IIα inhibitor, caused apoptosis. However, cells pretreated with Ang II were resistant to this apoptosis. Ang II significantly reduced the ratio of apoptotic cells and stimulation of phospho-Akt-Thr308 and phospho-Akt-Ser473 in a dose-dependent and time-dependent manner. In addition, Ang II significantly prevented apoptosis through inhibiting the cleavage of procaspase-9, a major downstream effector of Akt. TheAng II type 1 receptor (AT1R) was responsible for these effects. Among the signaling molecules downstream of AT1R, we revealed that the phosphatidylinositol 3-kinase/Akt pathway plays a predominant role in the anti-apoptotic effect of Ang II. Our data indicated that Ang n plays a critical anti-apoptotic role in breast cancer cells by a mechanism involving AT1R/phosphatidylinositol 3-kinase/Akt activation and the subsequent suppression of caspase-9 activation.     

Angiotensin and calcium signaling in the pituitary and hypothalamus

1) In the rat pituitary, angiotensin type 1B receptors (AT_1B) are located in lactotrophs and corticotrophs.2) Activation of AT_1B receptors are coupled to G_q/11 (Guanine protein coupled receptor, or GPCR); they increase phospholipase C (PLC) activity resulting in inositol 1,4,5 triphosphate (InsP₃) and diacylglycerol (DAG) formation. A biphasic increase in [Ca²⁺]_itriggered by InsP₃ and DAG ensues.3) As many GPCRs, AT_1B pituitary receptors rapidly desensitize.4) This was observed in the generation of InsP3, the mobilization of intracellular Ca²⁺, and in prolactin release. Both homologous and heterologous desensitization was evidenced.5) Desensitization of the angiotensin II type 1 (AT1) receptor in the pituitary shares similarities and differences with endogenously expressed or transfected AT1 receptors in different cell types.6) In the pituitary hyperplasia generated by chronic estrogen treatment there was desensitization or alteration in angiotensin II (Ang II) evoked intracellular Ca²⁺ increase, InsP3 generation, and prolactin release. This correlates with a downregulation of AT1 receptors.7) In particular, in hyperplastic cells Ang II failed to evoke a transient acute peak in [Ca²⁺]_i, which was replaced by a persistent plateau phase of [Ca²⁺]_i increase.8) Different calcium channels participate in Ang II induced [Ca²⁺]_i increase in control and hyperplastic cells. While spike phase in control cells is dependent on intracellular stores sensitive to thapsigargin, in hyperplastic cells plateau increase is dependent on extracellular calcium influx.9) Signal transduction of the AT1 pituitary receptor is greatly modified by hyperplasia, and it may be an important mechanism in the control of the hyperplastic process.10) In the hypothalamus and brain stem there is a predominant expression of AT_1A and AT₂ mRNA.11) Ang II acts at specific receptors located on neurons in the hypothalamus and brain stem to elicit alterations in blood pressure, fluid intake, and hormone secretion.12) Calcium channels play important roles in the Ang II induced behavioral and endocrine responses.13) Ang II, in physiological concentrations, can activate AT1 receptors to stimulate both Ca²⁺ release from intracellular stores and Ca²⁺ influx from the extracellular space to increase [Ca²⁺]_i in polygonal and stellate astroglia of the hypothalamus and brain stem.14) In primary cell culture of neurons from newborn rat hypothalamus and brain stem, it has also been determined that Ang II elicits an AT1 receptor mediated inhibition of delayed rectifier K(+) current and a stimulation of Ca²⁺ current.15) In primary cell cultures derived from the subfornical organ or the organum vasculosum laminae terminalis of newborn rat pups, Ang II produced a pronounced desensitization of the [Ca²⁺]_i response.16) Hypothalamic and pituitary Ang II systems are involved in different functions, some of which are related. At both levels Ang II signals through [Ca²⁺]_i in a characteristic way.     

The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone

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Angiotensin II receptors are reduced in the CNS of the young Brattleboro rat

In the rat, angiotensin II (AII), following specific interaction with sensitive central nervous system (CNS) receptors promotes release of vasopressin (ADH). We have examined the integrity of this chain of events by comparing the concentration, Bmax, and dissociation constant, Kd, in the CNS of Brattleboro rats (BB), a strain incapable of synthesizing ADH, with Long Evans (LE) control rats that can synthesize ADH. AII binding properties in the hypothalamic-thalamic-septal-midbrain (HTSM) area from young and old BB and LE, as well as systolic blood pressure, were determined. There was a reduction in the HTSM-AII receptor concentration of young BB when compared with young LE rats. Young BB had lower pressure than age and sex matched LE controls. Neither Bmax nor pressure was significantly different between older BB and LE. A decline in HTSM-AII receptor concentration with age observed with LE is consistent with observations in SHR and WKY rats. Parallel Scatchard plots obtained indicated the presence of a single class of CNS AII receptors. These data suggest that ADH synthesis and AII receptor concentration are partially interdependent and that the CNS AII-ADH system is redundant in the maintenance of blood pressure.     

Angiotensin II induces diverse signal transduction pathways via both Gq and Gi proteins in liver epithelial cells

Angiotensin II stimulates a biphasic activation of Raf-1, MEK, and ERK in WB liver epithelial cells. The first peak of activity is rapid and transient and is followed by a sustained phase. Angiotensin II also causes a rapid activation of p21^ras in these cells. Moreover, two Src family kinases (Fyn and Yes) were activated by angiotensin II in a time- and concentration-dependent manner. Microinjection of antibodies against Fyn and Yes blocked angiotensin II-induced DNA synthesis and c-Fos expression in WB cells, indicating an obligatory involvement of these tyrosine kinases in the activation of the ERK cascade by angiotensin II. Finally, substantial reduction of the angiotensin II-stimulated activation of Fyn, Raf-1, ERK, and expression of c-Fos by pertussis toxin pretreatment argues that G proteins of the G_i family as well as the G_q family are involved in angiotensin II-mediated mitogenic pathways in WB cells. J. Cell. Biochem. 69:63–71, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Angiotensin II induces endothelial cell senescence via the activation of mitogen-activated protein kinases

Vascular endothelial cells have a finite cell lifespan and eventually enter an irreversible growth arrest, cellular senescence. The functional changes associated with cellular senescence are thought to contribute to human aging and age-related cardiovascular disorders, for example, atherosclerosis. Angiotensin II (Ang II), a principal effector of the renin-angiotensin system (RAS), an important signaling molecule involved in atherogenic stimuli, is known to promote aging and cellular senescence. In the present study, induction of Ang II promoted a growth arrest with phenotypic characteristics of cell senescence, such as enlarged cell shapes, increased senescence-associated beta-galactosidase (SA-beta-gal) positive staining cells, and depressed cell proliferation. Ang II drastically decreased the expression level of Bcl-2, in part via the activation of extracellular signal-regulated kinase (ERK). Our results suggest that Ang II can induce HUVEC senescence; one of its molecular mechanisms is a probability that the mitogen-activated protein kinase (MAPK) signal pathway is involved in the process of pathological and physiological senescence of endothelial cells as well as vascular aging.     

Species Differences in Angiotensin II Generation and Degradation by Mast Cell Chymases

Abstract

Although chymases are known to exhibit species differences in regard to angiotensin (Ang) II generation and degradation, their properties have never been compared under the same experimental conditions. We analyzed the processing of Ang I by chymases of a variety of species (human chymase, dog chymase, hamster chymase-1, rat mast cell protease-1 [rMCP-1], mouse mast cell protease-4 [mMCP-4]) at physiological ionic strength and under neutral pH conditions. Human chymase generated Ang II from Ang I without further degradation, whereas the chymases of other species generated Ang II, followed by degradation at the Tyr⁴-Ile⁵ site in a time-dependent manner. Kinetic analysis showed that in terms of Ang II generating activity (analyzed by cleavage of the Phe⁸-His⁹ bond using the model peptide Ang, Ile⁵-His⁶-Pro⁷-Phe⁸-His⁹-Leu¹⁰), the chymases ranked as follows:dog > human > hamster > mouse > rat (k_cat/K_m: 18, 11, 0.69, 0.059, 0.030 μ M^{? 1}min^{? 1}), and that in terms of Ang II degrading activity (i.e., cleavage of the Tyr⁴-Ile⁵ bond of Ang II), the order was hamster > rat > mouse > dog (k_cat/K_m: 5.4, 4.8, 0.39, 0.29 μ M^?1min^?1). These results suggest species differences in the contribution of chymases to local Ang II generation and degradation.     

Angiotensin receptor II is present in dopaminergic cell line of rat substantia nigra and it is down regulated by aminochrome

Angiotensin receptor II mRNA was found to be expressed in dopaminergic neuronal cell line RCSN3 of rat substantia nigra using RT-PCR reaction. Aminochrome (150 M), a metabolite of the dopamine oxidative pathway, was found to down regulate the expression of angiotensin receptor mRNA in RCSN3 cells by 83% (p < 0.05).     

Effect of transient overexpression of Gqα on soluble and polymerized tubulin pools in GH3 and AtT-20 cells

In order to study G_q-tubulin interaction in the cytosol, GH₃ and AtT-20 cells (stably expressing TRH receptor) were transiently transfected with G_qα cDNA. Forty-eight hours after transfection, thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion by G_qα-transfected GH₃ cells increased by 90% compared to mock-transfected cells. In addition, using immunocytochemistry it was observed that G_qα-specific staining was much more prominent in G_qα-transfected GH₃ and AtT-20 cells (also transfected with G_qα) compared to mock-transfected cells. Thus, transfection resulted in successful overexpression of functional G_qα. Forty-eight hours after transfection, cells were processed to obtain soluble and polymerized tubulin fractions. Tubulin levels were determined in these fractions by immunoblotting using polyclonal anti-tubulin antibodies. Compared to mock-transfected cells soluble tubulin levels decreased in G_qα-transfected GH₁ and AtT-20 cells, by 33 and 52%, respectively. Moreover, compared to mock-transfected cells a 50% reduction in the ratio (an index of the flux between tubulin pools) of soluble and polymerized tubulin levels was observed in G_qα-transfected GH₃ and AtT-20 cells. To determine whether these effects on tubulin were mediated by G_q directly, we examined the influence of purified G_q on tubulin polymerization. G_q (0.5 μM) inhibited polymerization of crude tubulin (present in GH₃ cell cytosol) by 53%. In contrast to its effects on GH₃ cell cytosol tubulin, G_q stimulated purified tubulin polymerization by 160%. These results suggest that G_q modulates the polymerization and depolymerization cycles of tubulin and that this modulation is in turn influenced by other unknown cellular components. © 1996 Wiley-Liss, Inc.     

A truncated isoform of pyroglutamyl aminopeptidase II produced by exon extension has dominant-negative activity

Thyrotropin-releasing hormone is inactivated in the extracellular space by a membrane-bound peptidase, pyroglutamyl aminopeptidase II (PPII), a member of the M1 family of zinc metallopeptidases. The functional significance of multiple PPII RNA species expression is unknown. We detected, in rat tissues, a RNA species derived from an alternative processing at the exon 14-intron 14 boundary. The alternatively processed RNA encoded a shorter version of PPII (PPII*), lacking part of the C-terminal domain. PPII* was expressed in COS-7 (or C6 glioma) cells but it did not exhibit any PPII activity. Co-transfection of PPII and increasing amounts of PPII* expression vectors resulted in a dose-dependent reduction in PPII activity and the formation of covalent PPII-PPII* heterodimers. PPII* is therefore a powerful dominant-negative isoform of PPII, and heterodimerization may be its mechanism of action. Natural expression of shortened versions of M1 aminopeptidases may constitute a new mode of regulation of their activity.     

Angiotensin II Inhibits Satellite Cell Proliferation and Prevents Skeletal Muscle Regeneration

Cachexia is a serious complication of many chronic diseases, such as congestive heart failure (CHF) and chronic kidney disease (CKD). Although patients with advanced CHF or CKD often have increased angiotensin II (Ang II) levels and cachexia and Ang II causes skeletal muscle wasting in rodents, the potential effects of Ang II on muscle regeneration are unknown. Muscle regeneration is highly dependent on the ability of a pool of muscle stem cells (satellite cells) to proliferate and to repair damaged myofibers or form new myofibers. Here we show that Ang II reduced skeletal muscle regeneration via inhibition of satellite cell (SC) proliferation. Ang II reduced the number of regenerating myofibers and decreased expression of SC proliferation/differentiation markers (MyoD, myogenin, and active-Notch) after cardiotoxin-induced muscle injury in vivo and in SCs cultured in vitro. Ang II depleted the basal pool of SCs, as detected in Myf5^nLacZ/+ mice and by FACS sorting, and this effect was inhibited by Ang II AT1 receptor (AT1R) blockade and in AT1aR-null mice. AT1R was highly expressed in SCs, and Notch activation abrogated the AT1R-mediated antiproliferative effect of Ang II in cultured SCs. In mice that developed CHF postmyocardial infarction, there was skeletal muscle wasting and reduced SC numbers that were inhibited by AT1R blockade. Ang II inhibition of skeletal muscle regeneration via AT1 receptor-dependent suppression of SC Notch and MyoD signaling and proliferation is likely to play an important role in mechanisms leading to cachexia in chronic disease states such as CHF and CKD.     

Angiotensin II induced catabolic effect and muscle atrophy are redox dependent

Angiotensin II (Ang II) causes skeletal muscle wasting via an increase in muscle catabolism. To determine whether the wasting effects of Ang II were related to its ability to increase NADPH oxidase-derived reactive oxygen species (ROS) we infused wild-type C57BL/6J or p47^phox−/− mice with vehicle or Ang II for 7 days. Superoxide production was increased 2.4-fold in the skeletal muscle of Ang II infused mice, and this increase was prevented in p47^phox−/− mice. Apocynin treatment prevented Ang II-induced superoxide production in skeletal muscle, consistent with Ang II increasing NADPH oxidase derived ROS. Ang II induced loss of body and skeletal muscle weight in C57BL/6J mice, whereas the reduction was significantly attenuated in p47^phox−/− animals. The reduction of skeletal muscle weight caused by Ang II was associated with an increase of proteasome activity, and this increase was completely prevented in the skeletal muscle of p47^phox−/− mice. In conclusion, Ang II-induced skeletal muscle wasting is in part dependent on NADPH oxidase derived ROS.     

Loss of Timp3 Gene Leads to Abdominal Aortic Aneurysm Formation in Response to Angiotensin II

Aortic aneurysm is dilation of the aorta primarily due to degradation of the aortic wall extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs), the proteases that degrade the ECM. Timp3 is the only ECM-bound Timp, and its levels are altered in the aorta from patients with abdominal aortic aneurysm (AAA). We investigated the causal role of Timp3 in AAA formation. Infusion of angiotensin II (Ang II) using micro-osmotic (Alzet) pumps in Timp3^−/− male mice, but not in wild type control mice, led to adverse remodeling of the abdominal aorta, reduced collagen and elastin proteins but not mRNA, and elevated proteolytic activities, suggesting excess protein degradation within 2 weeks that led to formation of AAA by 4 weeks. Intriguingly, despite early up-regulation of MMP2 in Timp3^−/−Ang II aortas, additional deletion of Mmp2 in these mice (Timp3^−/−/Mmp2^−/−) resulted in exacerbated AAA, compromised survival due to aortic rupture, and inflammation in the abdominal aorta. Reconstitution of WT bone marrow in Timp3^−/−/Mmp2^−/− mice reduced inflammation and prevented AAA in these animals following Ang II infusion. Treatment with a broad spectrum MMP inhibitor (PD166793) prevented the Ang II-induced AAA in Timp3^−/− and Timp3^−/−/Mmp2^−/− mice. Our study demonstrates that the regulatory function of TIMP3 is critical in preventing adverse vascular remodeling and AAA. Hence, replenishing TIMP3, a physiological inhibitor of a number of metalloproteinases, could serve as a therapeutic approach in limiting AAA development or expansion.