Renal functional effects of the highly selective AT2R agonist, ß-Pro7 Ang III, in normotensive rats.

Recently, we designed a group of peptides by sequential substitution of the naturally occurring α-amino acid throughout the Ang III peptide sequence with the corresponding ß-amino acid. ß-Amino acid substitution at the proline residue of Ang III (ß-Pro7-Ang III) resulted in a highly selective AT2R ligand, demonstrating remarkable selectivity for the AT2R in both binding and functional studies. To provide additional functional evidence for the suitability of ß-Pro7 Ang III as a novel AT2R agonist, we tested effects of acute systemic administration of ß-Pro7-Ang III on renal hemodynamic and excretory function in anesthetized normotensive male and female rats. We also compared the natriuretic effects of acute intrarenal administration of native Ang III and ß-Pro7-Ang III in the presence of systemic AT1R blockade in anesthetized female rats to allow for the differentiation of systemic versus direct intrarenal natriuretic actions of ß-Pro7-Ang III. In both male and female rats, acute systemic administration of ß-Pro7-Ang III elicited renal vasodilatation and natriuresis. Notably, greater renal vasodilatory effects were observed in female versus male rats at the highest dose of ß-Pro7-Ang III administered. Moreover, intra-renal administration of ß-Pro7-Ang III produced significant natriuretic effects in female rats and, like Ang III, evoked AT2R translocation to the apical plasma membrane in renal proximal tubular cells. Taken together, our findings support the use of ß-Pro7-Ang III as a novel AT2R agonist and experimental tool for exploring AT2R function and its potential as a therapeutic target. Furthermore, our findings provide further evidence of a sex-specific influence of AT2R stimulation on renal function.

Effects of angiotensin III on c-Jun N terminal kinase in Wistar and hypertensive rat vascular smooth muscle cells.

Proliferation of vascular smooth muscle cells (VSMCs) and inflammation are well known actions associated with hypertension. Angiotensin (Ang) II mediates these physiological actions through the c-Jun N terminal Kinase (JNK), mitogen-activated proteins kinase (MAPK) pathway. Ang III effects on this pathway in VSMCs are unknown. The aim of this study was to determine whether Ang III activates JNK MAPK in Wistar VSMCs and determined whether the response was different in spontaneously hypertensive rat (SHR) VSMCs. We also ascertained whether this effect leads to VSMC proliferation. Western blots were used to determine the time and concentration effects of Ang II on JNK MAPK phosphorylation in Wistar VSMCs. Similar studies were conducted for Ang III in Wistar and SHR VSMCs. Both peptides induced JNK phosphorylation in a concentration- and time-dependent manner in Wistar VSMCs. Ang III also increased JNK phosphorylation in a concentration- and time-dependent fashion in SHR VSMCs as well. However, the ability of Ang III to induce JNK MAPK was different in SHR VSMCs as the phosphorylation levels of JNK were significantly higher in Wistar VSMCs as compared to SHR VSMCs at several time points and concentrations. Further, Ang III-mediated DNA synthesis, a measure of VSMC proliferation, occurred through activation of JNK MAPK. This study is the first to show Ang III effects on the JNK MAPK pathway in VSMCs and the role of JNK in Ang III-mediated cellular proliferation. These findings impart key information for the understanding of Ang III functions, especially in VSMCs and possible cardiovascular diseases.

Angiotensin III Induces JAK2/STAT3 Leading to IL-6 Production in Rat Vascular Smooth Muscle Cells.

The Janus kinase-2/ signal transducer and activators of transcription-3 (JAK2/STAT3) pathway and interleukin-6 (IL-6) are pleiotropic signal transduction systems that are responsible for induction of many cytokines and growth factors. It is unknown whether the renin angiotensin aldosterone system (RAAS) peptide, angiotensin (Ang) III induces JAK2/STAT3 and IL-6 in vascular smooth muscle cells (VSMCs). Thus, the purpose of this study was to investigate whether Ang III induces the JAK2/STAT3 pathway leading to IL-6 production in cultured VSMCs isolated from Wistar rats and determine whether differences exist in spontaneously hypertensive rat (SHR) VSMCs. We gauged Ang III's effects on this pathway by measuring its action on STAT3 as well as IL-6 production. Ang III behaved similarly as Ang II in stimulation of STAT3 phosphorylation in Wistar and SHR VSMCs. Moreover, there were no differences in this Ang III effect in SHR versus Wistar VSMCs. In Wistar VSMCs, Ang II and Ang III significantly induced IL-6 protein secretion and mRNA expression. However, IL-6 protein secretions mediated by these peptides were significantly greater in SHR VSMCs. Ang III induced the JAK2/STAT3 pathway, leading to IL-6 protein secretion and IL-6 mRNA expression via actions on AT1Rs. Moreover, the actions of Ang III to induce IL-6 production was dysregulated in SHR VSMCs. These findings suggest that Ang III acts on AT1Rs to induce JAK2/STAT3, leading to an increase in IL-6 in cultured VSMCs. These findings are important in establishing Ang III as an important physiologically relevant peptide in VSMCs.

Angiotensin III increases monocyte chemoattractant protein-1 expression in cultured human proximal tubular epithelial cells.

BACKGROUND/AIMS: We investigated whether angiotensin III (Ang III) is involved in monocyte recruitment through regulation of the chemokine monocyte chemoattractant protein-1 (MCP-1) in cultured human proximal tubular epithelial cells (HK-2 cells). METHODS: We measured MCP-1 levels in HK-2 cells that had been treated with various concentrations of Ang III and Ang II type-1 (AT1) receptor antagonists at various time points. The phosphorylation states of p38, c-Jun N-terminal kinases (JNK), and extracellular-signal-regulated kinases were measured in Ang III-treated cells to explore the mitogen-activated protein kinase (MAPK) pathway. MCP-1 levels in HK-2 cell-conditioned media were measured after pre-treatment with the transcription factor inhibitors curcumin or pyrrolidine dithiocarbamate. RESULTS: Ang III increased MCP-1 protein production in dose- and time-dependent manners in HK-2 cells, which was inhibited by the AT1 receptor blocker losartan. p38 MAPK activity increased significantly in HK-2 cells exposed to Ang III for 30 minutes, and was sustained at higher levels after 60 minutes (p < 0.05). Total phosphorylated JNK protein levels tended to increase 20 minutes after stimulation with Ang III. Pre-treatment with a p38 inhibitor, a JNK inhibitor, or curcumin significantly inhibited Ang III-induced MCP-1 production. CONCLUSIONS: Ang III increases MCP-1 synthesis via stimulation of intracellular p38 and JNK MAPK signaling activity and subsequent activated protein-1 transcriptional activity in HK-2 cells.

Angiotensin III increases monocyte chemoattractant protein-1 expression in cultured human proximal tubular epithelial cells

BACKGROUND/AIMS: We investigated whether angiotensin III (Ang III) is involved in monocyte recruitment through regulation of the chemokine monocyte chemoattractant protein-1 (MCP-1) in cultured human proximal tubular epithelial cells (HK-2 cells). METHODS: We measured MCP-1 levels in HK-2 cells that had been treated with various concentrations of Ang III and Ang II type-1 (AT1) receptor antagonists at various time points. The phosphorylation states of p38, c-Jun N-terminal kinases (JNK), and extracellular-signal-regulated kinases were measured in Ang III-treated cells to explore the mitogen-activated protein kinase (MAPK) pathway. MCP-1 levels in HK-2 cell-conditioned media were measured after pre-treatment with the transcription factor inhibitors curcumin or pyrrolidine dithiocarbamate. RESULTS: Ang III increased MCP-1 protein production in dose- and time-dependent manners in HK-2 cells, which was inhibited by the AT1 receptor blocker losartan. p38 MAPK activity increased significantly in HK-2 cells exposed to Ang III for 30 minutes, and was sustained at higher levels after 60 minutes (p < 0.05). Total phosphorylated JNK protein levels tended to increase 20 minutes after stimulation with Ang III. Pre-treatment with a p38 inhibitor, a JNK inhibitor, or curcumin significantly inhibited Ang III-induced MCP-1 production. CONCLUSIONS: Ang III increases MCP-1 synthesis via stimulation of intracellular p38 and JNK MAPK signaling activity and subsequent activated protein-1 transcriptional activity in HK-2 cells.

ß-Pro7Ang III is a novel highly selective angiotensin II type 2 receptor (AT2R) agonist, which acts as a vasodepressor agent via the AT2R in conscious spontaneously hypertensive rats.

We have previously shown that individual ß-amino acid substitution in angiotensin (Ang) II reduced Ang II type 1 receptor (AT1R) but not Ang II type 2 receptor (AT2R)-binding and that the heptapeptide Ang III exhibited greater AT2R:AT1R selectivity than Ang II. Therefore, we hypothesized that ß-amino-acid-substituted Ang III peptide analogues would yield highly selective AT2R ligands, which we have tested in binding and functional vascular assays. In competition binding experiments using either AT1R- or AT2R-transfected human embryonic kidney (HEK)-293 cells, novel ß-substituted Ang III analogues lacked appreciable AT1R affinity, whereas most compounds could fully displace (125)I-Sar(1)Ile(8) Ang II from AT2R. The rank order of affinity at AT2R was CGP42112 > Ang III > ß-Pro(7) Ang III=Ang II > ß-Tyr(4) Ang III ≥ PD123319 >> ß-Phe(8) Ang III >> ß Arg(2) Ang III=ß-Val(3) Ang III >> ß-Ile(5) Ang III. The novel analogue ß-Pro(7) Ang III was the most selective AT2R ligand tested, which was >20,000-fold more selective for AT2R than AT1R. IC50 values at AT2R from binding studies correlated with maximum vasorelaxation in mouse aortic rings. Given that ß-Pro(7) Ang III was an AT2R agonist, we compared ß-Pro(7) Ang III and native Ang III for their ability to reduce blood pressure in separate groups of conscious spontaneously hypertensive rats. Whereas Ang III alone increased mean arterial pressure (MAP), ß-Pro(7) Ang III had no effect. During low-level AT1R blockade, both Ang III and ß-Pro(7) Ang III, but not Ang II, lowered MAP (by ∼30 mmHg) at equimolar infusions (150 pmol/kg/min for 4 h) and these depressor effects were abolished by the co-administration of the AT2R antagonist PD123319. Thus, ß-Pro(7) Ang III has remarkable AT2R selectivity determined in binding and functional studies and will be a valuable research tool for insight into AT2R function and for future drug development.

Angiotensin III induces signal transducer and activator of transcription 3 and interleukin-6 mRNA levels in cultured rat astrocytes.

INTRODUCTION: Recently we established that pro-inflammatory actions of angiotensin (Ang) II in astrocytes involved Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and interleukin-6 (IL-6). MATERIALS AND METHODS: In our current study, we determined in brainstem and cerebellum whether Ang III also activates STAT3 leading to IL-6 mRNA expression and astrocyte proliferation. RESULTS: Ang III induced STAT3 phosphorylation in a concentration- and time-dependent manner. Significant STAT3 phosphorylation was rapid and was maximal within 10 min, and with 100 nM Ang III. The Ang AT1 receptor was shown to mediate this action of Ang III. Ang III also significantly induced IL-6 mRNA expression within an hour, and maximal Ang III-mediated IL-6 mRNA expression occurred in the presence of 100 nM Ang III. Ang III-mediated IL-6 mRNA expression occurred by the interaction of the peptide with the Ang AT1 receptor and was mediated by STAT3. In addition, STAT3 was shown to mediate Ang III astrocyte proliferation. CONCLUSIONS: These findings suggest that Ang III, similar to Ang II, has pro-inflammatory effects since it induces STAT3 leading to an induction of IL-6 mRNA expression, outcomes that lend relevance to the physiological importance of central Ang III.

Angiotensin III stimulates high stretch-induced ANP secretion via angiotensin type 2 receptor.

Angiotensin III (Ang III) is metabolized from Ang II by aminopeptidase (AP) A and in turn, Ang III is metabolized to Ang IV by APN. Ang III is known to have a similar effect to Ang II on aldosterone secretion, but the effect of Ang III on atrial natriuretic peptide (ANP) secretion from cardiac atria is not known. The aim of the present study is to define the effect of Ang III on ANP secretion and its receptor subtype using isolated perfused beating atria. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH2O to 7.5 cmH2O. Atrial stretch by volume load increased atrial contractility and ANP secretion. Ang III stimulated stretch-induced ANP secretion in a dose-dependent manner without change in atrial contractility. The stimulated effect of Ang III (1 µM) on stretch-induced ANP secretion was blocked by the pretreatment of Ang II type 2 (AT2) receptor antagonist but not by AT1 or Mas receptor antagonist. Pretreatment with inhibitor of phosphoinositide 3-kinase (PI3K), Akt, nitric oxide synthase, soluble guanylyl cyclase, or protein kinase G (PKG) attenuated Ang III-stimulated ANP secretion. When Ang III (40 nM) or Ang II (4nM) was infused for 10 min into anesthetized rats, mean arterial pressure was increased about 10%. However, Ang III increased plasma ANP level by 35.81±10.19% but Ang II decreased plasma ANP level by 30.41±7.27%. Therefore, we suggest that Ang III, opposite to Ang II, stimulated stretch-induced ANP secretion through AT2 receptor/PI3K/Akt/nitric oxide/PKG pathway.

Angiotensin II and III metabolism and effects on steroid production in the HAC15 human adrenocortical cell line.

Aldosterone is synthesized in the zona glomerulosa of the adrenal cortex under primary regulation by the renin-angiotensin system. Angiotensin II (A-II) acts through the angiotensin types 1 and 2 receptors (AT1R and AT2R). A-II is metabolized in different tissues by various enzymes to generate two heptapeptides A-III and angiotensin 1-7, which can then be catabolized into smaller peptides. A-II was more potent than A-III in stimulating aldosterone secretion in the adrenocortical cell line HAC15, and A-II, but not A-III, stimulated cortisol secretion. A-II stimulated mRNA expression of steroidogenic acute regulatory protein, 3ß-hydroxysteroid dehydrogenase, CYP11B1, and CYP11B2, whereas A-III stimulated 3ß-hydroxysteroid dehydrogenase, CYP11B1, and CYP11B2 but decreased the expression of CYP17A1 required for cortisol synthesis. The stimulation of aldosterone secretion by A-II and A-III was blocked by the AT1R receptor blocker, losartan, but not by an AT2R blocker. A-II was rapidly metabolized by the HAC15 cells to mainly to angiotensin 1-7, but not to A-III, and disappeared from the supernatant within 6 h. A-III was metabolized rapidly and disappeared within 1 h. In conclusion, A-II was not converted to A-III in the HAC15 cell and is the more potent stimulator of aldosterone secretion and cortisol of the two. A-III stimulated aldosterone secretion but not cortisol secretion.

Similarities and differences between effects of angiotensin III and angiotensin II on human prostate cancer cell migration and proliferation.

Proliferation plays a critical role in tumor growth when cell migration is essential to invasion. The effect of Ang III and Ang II was evaluated on these important processes. Changes in the migration potential of prostate cancer cells were investigated using Wound Healing Test and a Transwell Migration Chamber with a 3 µm pore size. Cell proliferation was measured with a BrdU Assay and Countess Automated Cell Counter, thus determining the influence of angiotensins on hormone-dependent (LNCaP) and hormone-independent (DU-145) human prostate cancer lines. The influence of Ang III and Ang II on classic receptors may be inhibited by Losartan or PD123319. Test peptide modulation of the AT1 and AT2 receptors was examined by Western Blot and fluorescent immunocytochemistry. The results indicate that Ang III promotes the migration of both LNCaP and DU-145 lines, whereas Ang II stimulates this process only in androgen-independent cells. Both angiotensin peptides can induce prostate cancer cell proliferation in a time- and dose-dependent manner. The obtained results show that Ang III and Ang II can modify the expression of classic receptors, particularly AT2. These results suggest that the investigated peptide can modulate cell migration and proliferation in prostate cancer cells. Angiotensins probably have a greater influence on proliferation in the early-stage prostate cancer model than hormone-independent cell lines. Assume also that Ang II can enhance the migration tendency aggressive prostate cancer cells, while Ang III does so more effective in non-metastatic cells.

Intrarenal angiotensin III is the predominant agonist for proximal tubule angiotensin type 2 receptors.

In angiotensin type 1 receptor-blocked rats, renal interstitial (RI) administration of des-aspartyl(1)-angiotensin II (Ang III) but not angiotensin II induces natriuresis via activation of angiotensin type 2 receptors. In the present study, renal function was documented during systemic angiotensin type 1 receptor blockade with candesartan in Sprague-Dawley rats receiving unilateral RI infusion of Ang III. Ang III increased urine sodium excretion, fractional sodium, and lithium excretion. RI coinfusion of specific angiotensin type 2 receptor antagonist PD-123319 abolished Ang III-induced natriuresis. The natriuretic response observed with RI Ang III was not reproducible with RI angiotensin (1-7) alone or together with angiotensin-converting enzyme inhibition. Similarly, neither RI angiotensin II alone or in the presence of aminopeptidase A inhibitor increased urine sodium excretion. In the absence of systemic angiotensin type 1 receptor blockade, Ang III alone did not increase urine sodium excretion, but natriuresis was enabled by the coinfusion of aminopeptidase N inhibitor and subsequently blocked by PD-123319. In angiotensin type 1 receptor-blocked rats, RI administration of aminopeptidase N inhibitor alone also induced natriuresis that was abolished by PD-123319. Ang III-induced natriuresis was accompanied by increased RI cGMP levels and was abolished by inhibition of soluble guanylyl cyclase. RI and renal tissue Ang III levels increased in response to Ang III infusion and were augmented by aminopeptidase N inhibition. These data demonstrate that endogenous intrarenal Ang III but not angiotensin II or angiotensin (1-7) induces natriuresis via activation of angiotensin type 2 receptors in the proximal tubule via a cGMP-dependent mechanism and suggest aminopeptidase N inhibition as a potential therapeutic target in hypertension.

Central ventilatory and cardiovascular actions of angiotensin peptides in trout.

In the brains of teleosts, angiotensin II (ANG II), one of the main effector peptides of the renin-angiotensin system, is implicated in various physiological functions notably body fluid and electrolyte homeostasis and cardiovascular regulation, but nothing is known regarding the potential action of ANG II and other angiotensin derivatives on ventilation. Consequently, the goal of the present study was to determine possible ventilatory and cardiovascular effects of intracerebroventricular injection of picomole doses (5-100 pmol) of trout [Asn(1)]-ANG II, [Asp(1)]-ANG II, ANG III, ANG IV, and ANG 1-7 into the third ventricle of unanesthetized trout. The central actions of these peptides were also compared with their ventilatory and cardiovascular actions when injected peripherally. Finally, we examined the presence of [Asn(1)]-ANG II, [Asp(1)]-ANG II, ANG III, and ANG IV in the brain and plasma using radioimmunoassay coupled with high-performance liquid chromatography. After intracerebroventricular injection, [Asn(1)]-ANG II and [Asp(1)]-ANG II two ANG IIs, elevated the total ventilation through a selective stimulatory action on the ventilation amplitude. However, the hyperventilatory effect of [Asn(1)]-ANG II was threefold higher than the effect of [Asp(1)]-ANG II at the 50-pmol dose. ANG III, ANG IV, and ANG 1-7 were without effect. In addition, ANG IIs and ANG III increased dorsal aortic blood pressure (P(DA)) and heart rate (HR). After intra-arterial injections, none of the ANG II peptides affected the ventilation but [Asn(1)]-ANG II, [Asp(1)]-ANG II, and ANG III elevated P(DA) (50 pmol: +80%, +58% and +48%, respectively) without significant decrease in HR. In brain tissue, comparable amounts of [Asn(1)]-ANG II and [Asp(1)]-ANG II were detected (ca. 40 fmol/mg brain tissue), but ANG III was not detected, and the amount of ANG IV was about eightfold lower than the content of the ANG IIs. In plasma, ANG IIs were also the major angiotensins (ca. 110 fmol/ml plasma), while significant but lower amounts of ANG III and ANG IV were present in plasma. In conclusion, our study suggests that the two ANG II isoforms produced within the brain may act as a neurotransmitter and/or neuromodulator to regulate the cardioventilatory functions in trout. In the periphery, two ANG IIs and their COOH-terminal peptides may act as a circulating hormone preferentially involved in cardiovascular regulations.

Angiotensin III induces c-Jun N-terminal kinase leading to proliferation of rat astrocytes.

Previously, we showed in cultured rat astrocytes that angiotensin (Ang) III induced astrocyte proliferation and phosphorylation of ERK1/2 mitogen activated protein (MAP) kinases through interaction with the AT(1) receptor. In the current study, we determined whether the c-Jun N terminal kinase (JNK) MAP kinase pathway was similarly affected by the peptide in cultured brainstem astrocytes. Ang III induced JNK phosphorylation in a concentration- and time-dependent manner. Similar to ERK1/2 phosphorylation, maximal phosphorylation occurred with 100 nM Ang III and was apparent within a minute of exposure to the peptide. Peak effects were observed over a 5-15 min time range. Pretreatment of brainstem astrocytes with the JNK inhibitor, SP600125, prevented Ang III phosphorylation of JNK, as well as Ang III-mediated astrocyte growth. The selective AT(1) receptor antagonist, Losartan, prevented Ang III-induced JNK phosphorylation. Pretreatment of astrocytes with the AT(2) receptor blocker PD123319 was ineffective in preventing JNK phosphorylation by Ang III. Interestingly, both Ang II and Ang III induced JNK phosphorylation to a similar extent suggesting that the two peptides were equipotent in this effect. Our findings suggest that Ang III interacts with Ang AT(1) receptors to directly stimulate the JNK MAP kinase pathway leading to astrocyte growth. This study is the first to show that Ang III actions may involve the JNK MAP kinase pathway in astrocytes and provide key information that may lead to a better understanding of the functions of Ang III in the central nervous system, in particular in astrocytes.

Angiotensin III stimulates ERK1/2 mitogen-activated protein kinases and astrocyte growth in cultured rat astrocytes.

Angiotensin (Ang) III is a biologically active metabolite of Ang II with similar effects and receptor binding properties as Ang II. Most Ang III studies delineate physiological effects of the peptide but, the intracellular pathways leading to the actions are unknown and are a focus of these studies. We investigated in cultured brainstem and cerebellum rat astrocytes whether Ang III stimulates ERK1/2 mitogen activated protein (MAP) kinases and astrocyte growth. Ang III significantly stimulated ERK1/2 MAP kinases in a dose- and time-dependent manner. The maximal stimulation occurred with 100 nM Ang III (2.8±0.3 and 2.3±0.1-fold over basal, in brainstem and cerebellum astrocytes, respectively). This stimulation occurred as early as 1 min, and was sustained for at least 15 min. Moreover, inhibition of the ERK1/2 MAP kinase pathway by 10 µM PD98059 attenuated Ang III-induced ERK1/2 phosphorylation. Ang III induction of ERK1/2 occurred via stimulation of the Ang AT(1) receptor since pretreatment with 10 µM Losartan, a selective AT(1) receptor blocker, prevented Ang III-induced ERK1/2 phosphorylation. The selective AT(2) Ang receptor blocker PD123319 was ineffective. Comparable to Ang II, Ang III also stimulated astrocyte growth in a concentration-dependent manner, an effect that occurred via activation of the AT(1) receptor as well. These findings suggest that Ang III has similar effects as Ang II in astrocytes since it rapidly stimulates the phosphorylation of the ERK1/2 MAP kinases and induces astrocyte proliferation through activation of the AT(1) receptor. These studies are important in establishing signaling pathways for Ang III and provide validation of the central role of Ang III.

Blood pressure and renal hemodynamic effects of angiotensin fragments.

Angiotensin (Ang) II, the main effector peptide of the renin-Ang system, increases arterial blood pressure through Ang II type 1A (AT(1a)) receptor-dependent arterial vasoconstriction and by decreasing renal salt and water excretion through extrarenal and intrarenal mechanisms. AT(2) receptors are assumed to oppose these responses mediated by AT(1) receptors, thereby attenuating the pressor effects of Ang II. Nevertheless, a possible role of AT(2) receptors in the regulation of renal hemodynamics and sodium homeostasis remains to be unclear. Several other Ang fragments such as Ang III, Ang IV, Ang-(1-7) and Ang A have also been shown to display biological activity. In this review, we focus on the effects of these Ang on blood pressure, renal hemodynamics and sodium water handling, and discuss the receptors involved in these actions.

Angiotensin III stimulates aldosterone secretion from adrenal gland partially via angiotensin II type 2 receptor but not angiotensin II type 1 receptor.

Angiotensin II (Ang II) and Ang III stimulate aldosterone secretion by adrenal glomerulosa, but the angiotensin receptor subtypes involved and the effects of Ang IV and Ang (1-7) are not clear. In vitro, different angiotensins were added to rat adrenal glomerulosa, and aldosterone concentration in the medium was measured. Ang II-induced aldosterone release was blocked (30.3 ± 7.1%) by an Ang II type 2 receptor (AT2R) antagonist, PD123319. Candesartan, an Ang II type 1 receptor (AT1R) antagonist, also blocked Ang II-induced aldosterone release (42.9 ± 4.8%). Coadministration of candesartan and PD123319 almost abolished the Ang II-induced aldosterone release. A selective AT2R agonist, CGP42112, was used to confirm the effects of AT2R. CGP42112 increased aldosterone secretion, which was almost completely inhibited by PD123319. In addition to Ang II, Ang III also induced aldosterone release, which was not blocked by candesartan. However, PD123319 blocked 22.4 ± 10.5% of the Ang III-induced aldosterone secretion. Ang IV and Ang (1-7) did not induce adrenal aldosterone secretion. In vivo, both Ang II and Ang III infusion increased plasma aldosterone concentration, but only Ang II elevated blood pressure. Ang IV and Ang (1-7) infusion did not affect blood pressure or aldosterone concentration. In conclusion, this report showed for the first time that AT2R partially mediates Ang III-induced aldosterone release, but not AT1R. Also, over 60% of Ang III-induced aldosterone release may be independent of both AT1R and AT2R. Ang III and AT2R signaling may have a role in the pathophysiology of aldosterone breakthrough.

Effects of angiotensin III on protein, DNA, and collagen synthesis of neonatal cardiomyocytes and cardiac fibroblasts in vitro.

This study compared angiotensin II (Ang II) and angiotensin III (Ang III) for their effects on rat neonatal cardiomyocytes and cardiac fibroblasts in vitro and discussed the possible role of Ang III in the pathogenesis of cardiac remodeling. To do so, protein synthesis, cardiac fibroblast proliferation, collagen synthesis, and secretion in response to treatment with Ang III and Ang II were investigated. Protein synthesis rate was assessed by (3)H-Leucine ((3)H-Leu) incorporation; the content of DNA was defined by (3)H-thymidine ((3)H-TdR) incorporation; and collagen synthesis and secretion were assessed by ( 3)H-proline ((3)H-Pro) incorporation. In neonatal cardiomyocytes, Ang III stimulated protein synthesis in a concentration-dependent manner, whereas in neonatal cardiac fibroblasts, DNA synthesis as well as collagen synthesis and secretion were increased in a concentration-dependent manner. Treatment with captopril, selective aminopeptidase A (APA) inhibitor (EC33), or selective aminopeptidase N inhibitor (PC18) had no effect on these outcomes. Treatment with losartan significantly decreased the effects of Ang III, except for cardiomyocyte protein synthesis. Compared with Ang II, Ang III could stimulate cardiomyocyte protein synthesis, cardiac fibroblast proliferation, and collagen synthesis and secretion. Furthermore, 10(-7) mol/L Ang II but not Ang III significantly increased APA activity in both cardiomyocytes and fibroblasts. All these results show the bioactive effects of Ang III on myocardial cells and suggest that Ang III could be an important independent factor besides Ang II in the regulation of cardiac function and may affect the pathogenesis of cardiac remodeling.

Effects of angiotensin metabolites in the coronary vascular bed of the spontaneously hypertensive rat: loss of angiotensin II type 2 receptor-mediated vasodilation.

Because angiotensin (Ang) metabolites mediate functions independent of Ang II, we investigated their effects on coronary flow in spontaneously hypertensive rats (SHRs). Results were compared with those in the iliac artery and abdominal aorta and the coronary circulation of the Wistar rat. Ang II, III, and IV decreased coronary flow in SHRs and Wistar rats, with Ang III and IV being approximately 10 and approximately 1000 times less potent than Ang II. Ang-(1-7) decreased coronary flow at concentrations >1 micromol/L in SHRs. The Ang II type 1 receptor antagonist irbesartan blocked the effects of Ang II, III, and IV, whereas the Ang II type 2 receptor antagonist PD123319 blocked the effects of Ang-(1-7). The maximal Ang II- and III-induced decreases in coronary flow in SHRs were twice as large as those in Wistar rats. PD123319 enhanced the constrictor effects of Ang II and III in Wistar rats so that, in the presence of this drug, their effects were comparable to those in SHRs. In contrast, PD123319 did not alter the Ang II- and III-induced responses in SHRs and blocked the constrictor effect of Ang II in iliac arteries. Ang II type 2 receptor-mediated relaxation did not occur in iliac arteries and abdominal aortas, and the constrictor effects of Ang metabolites in these vessels were identical in Wistar rats and SHRs. In conclusion, coronary constriction induced by Ang II, Ang III, and Ang-(1-7) is enhanced in SHRs as compared with Wistar rats. This is attributable to the absence of counterregulatory Ang II type 2 receptor-mediated relaxation and/or a change of the Ang II type 2 receptor phenotype from relaxant to constrictor.

Angiotensin III modulates the nociceptive control mediated by the periaqueductal gray matter.

Endogenous angiotensin (Ang) II and/or an Ang II-derived peptide, acting on Ang type 1 (AT(1)) and Ang type 2 (AT(2)) receptors, can carry out part of the nociceptive control modulated by periaqueductal gray matter (PAG). However, neither the identity of this putative Ang-peptide, nor its relationship to Ang II antinociceptive activity was clarified. Therefore, we have used tail-flick and incision allodynia models combined with an HPLC time course of Ang metabolism, to study the Ang III antinociceptive effect in the rat ventrolateral (vl) PAG using peptidase inhibitors and receptor antagonists. Ang III injection into the vlPAG increased tail-flick latency, which was fully blocked by Losartan and CGP 42,112A, but not by divalinal-Ang IV, indicating that Ang III effect was mediated by AT(1) and AT(2) receptors, but not by the AT(4) receptor. Ang III injected into the vlPAG reduced incision allodynia. Incubation of Ang II with punches of vlPAG homogenate formed Ang III, Ang (1-7) and Ang IV. Amastatin (AM) inhibited the formation of Ang III from Ang II by homogenate, and blocked the antinociceptive activity of Ang II injection into vlPAG, suggesting that aminopeptidase A (APA) formed Ang III from Ang II. Ang III can also be formed from Ang I by a vlPAG alternative pathway. Therefore, the present work shows, for the first time, that: (i) Ang III, acting on AT(1) and AT(2) receptors, can elicit vlPAG-mediated antinociception, (ii) the conversion of Ang II to Ang III in the vlPAG is required to elicit antinociception, and (iii) the antinociceptive activity of endogenous Ang II in vlPAG can be ascribed preponderantly to Ang III.

Mammalian AT2 receptors expressed in Xenopus laevis oocytes couple to endogenous chloride channels and stimulate germinal vesicle break down.

BACKGROUND AND AIMS: A comparative analysis was performed of the properties of cloned AT1 and AT2 receptors and their ability to induce ion currents and oocyte maturation. METHODS: Frog oocytes were injected with cRNA that codes for rat AT1A and AT2 and bovine AT1 receptors and membrane currents were recorded by using the two-microelectrode voltage-clamp technique. Oocyte maturation was scored by observation of the germinal vesicle breakdown (GVBD). RESULTS: Xenopus oocytes expressing AT1 or AT2 generated oscillatory chloride currents by coupling to the diacylglycerol/inositol-3-phosphate (DAG/IP(3)) cascade. Ang II activation of collagenase-defolliculated oocytes expressing rat AT1A yielded larger chloride currents (9.2+/-3.4 A) than those generated by oocytes expressing bovine AT1 (3.78+/-2.6 A). Oocytes expressing rat AT1A were recovered from desensitization after 0.5 h and completely after 10.5 h; whereas oocytes expressing bovine AT1 receptors were unable to do so. Expression of rat AT2 generated smaller chloride currents (32-210 nA). Expression of AT2 receptors triggered GVBD whereas AT1 did not. The proportion of oocytes developing GVBD was greater for folliculated oocytes than for oocytes that were defolliculated. Furthermore, oocytes injected with AT2 secreted into the media, a heat-resistant factor(s) that stimulated GVBD of oocytes. This media elicited inward and outward membrane currents when applied to non-injected oocytes. CONCLUSION: Activation of AT1 and AT2 receptors couple to similar metabolic cascades in Xenopus laevis oocytes. However, the pathway activated by AT2 diverges to induce oocyte maturation.