Angiotensin II regulates vascular and endothelial dysfunction: recent topics of Angiotensin II type-1 receptor signaling in the vasculature

Accumulating evidence strongly implicates angiotensin II (AngII) intracellular signaling in mediating cardiovascular diseases such as hypertension, atherosclerosis and restenosis after vascular injury. In vascular smooth muscle cells (VSMCs), through its G-protein-coupled AngII Type 1 receptor (AT(1)), AngII activates various intracellular protein kinases, such as receptor or non-receptor tyrosine kinases, which includes epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), c-Src, PYK2, FAK, JAK2. In addition, AngII activates serine/threonine kinases such as mitogen-activated protein kinase (MAPK) family, p70 S6 kinase, Akt/protein kinase B and various protein kinase C isoforms. In VSMCs, AngII also induces the generation of intracellular reactive oxygen species (ROS), which play critical roles in activation and modulation of above signal transduction. Less is known about endothelial cell (EC) AngII signaling than VSMCs, however, recent studies suggest that endothelial AngII signaling negatively regulates the nitric oxide (NO) signaling pathway and thereby induces endothelial dysfunction. Moreover, in both VSMCs and ECs, AngII signaling cross-talk with insulin signaling might be involved in insulin resistance, an important risk factor in the development of cardiovascular diseases. In fact, clinical and pharmacological studies showed that AngII infusion induces insulin resistance and AngII converting enzyme inhibitors and AT(1) receptor blockers improve insulin sensitivity. In this review, we focus on the recent findings that suggest the existence of novel signaling mechanisms whereby AngII mediates processes, such as activation of receptor or non-receptor tyrosine kinases and ROS, as well as cross-talk between insulin and NO signal transduction in VSMCs and ECs.     

Biased agonism of the angiotensin II type 1 receptor

G protein-coupled receptors (GPCRs) can be activated by multiple ligands and exhibit the capacity to couple to numerous intracellular signal transduction pathways. This property allows GPCRs to be modulated by biased agonists that selectively activate specific subsets of GPCR-regulated cellular signaling proteins. The angiotensin II type 1 receptor (AT1R) is a GPCR that endogenously binds to the peptide ligand angiotensin II. More recently it has been demonstrated that a modified peptide, [Sar1I-le4-Ile8]-angiotensin II (SII) acts as a biased agonist towards the AT1R. SII binds to the AT1R without promoting heterotrimeric G protein-coupling, but serves to link the receptor to the beta-arrestin-dependent activation of the mitogen activated protein kinase pathway. The present mini-review summarizes current knowledge regarding the role of biased agonists in stimulating biased AT1R signaling.     

Side-chain substitutions within angiotensin II reveal different requirements for signaling, internalization, and phosphorylation of type 1A angiotensin receptors

Binding of the peptide hormone angiotensin II (AngII) to the type 1 (AT(1A)) receptor and the subsequent activation of phospholipase C-mediated signaling, involves specific determinants within the AngII peptide sequence. In contrast, the contribution of such determinants to AT(1A) receptor internalization, phosphorylation and activation of mitogen-activated protein kinase (MAPK) signaling is not known. In this study, the internalization of an enhanced green fluorescent protein-tagged AT(1A) receptor (AT(1A)-EGFP), in response to AngII and a series of substituted analogs, was visualized and quantified using confocal microscopy. AngII-stimulation resulted in a rapid, concentration-dependent internalization of the chimeric receptor, which was prevented by pretreatment with the nonpeptide AT(1) receptor antagonist EXP3174. Remarkably, AT(1A) receptor internalization was unaffected by substitution of AngII side chains, including single and double substitutions of Tyr(4) and Phe(8) that abolish phospholipase C signaling through the receptor. AngII-induced receptor phosphorylation was significantly inhibited by several substitutions at Phe(8) as well as alanine replacement of Asp(1). The activation of MAPK was only significantly inhibited by substitutions at position eight in the peptide and specific substitutions did not equally inhibit inositol phosphate production, receptor phosphorylation and MAPK activation. These results indicate that separate, yet overlapping, contacts made between the AngII peptide and the AT(1A) receptor select/induce distinct receptor conformations that preferentially affect particular receptor outcomes. The requirements for AT(1A) receptor internalization seem to be less stringent than receptor activation and signaling, suggesting an inherent bias toward receptor deactivation.     

AT1 receptor blocker-insensitive mutant AT1A angiotensin receptors reveal the presence of G protein-independent signaling in C9 cells

Although mutant receptors are highly useful to dissect the signal transduction pathways of receptors, they are difficult to study in physiological target tissues, due to the presence of endogenous receptors. To study AT(1) angiotensin receptors in their physiological environment, we constructed a mutant receptor, which differs only from the AT(1A) receptor in its reduced affinity for candesartan, a biphenylimidazole antagonist. We have determined that the conserved S109Y substitution of the rat AT(1A) receptor eliminates its candesartan binding, without exerting any major effect on its angiotensin II and peptide angiotensin receptor antagonist binding, internalization kinetics, beta-arrestin binding, and potency or efficacy of the inositol phosphate response. To demonstrate the usefulness of this mutant receptor in signal transduction studies, we combined it with substitution of the highly conserved DRY sequence with AAY, which abolishes G protein activation. In rat C9 hepatocytes the S109Y receptor caused ERK activation with the same mechanism as the endogenous AT(1) receptor. After combination with the DRY/AAY mutation G protein-independent ERK activation was detected demonstrating that this approach can be used to study the angiotensin II-stimulated signaling pathways in cells endogenously expressing AT(1) receptors.     

Hybrid formation between the intracellular faces of the bradykinin B2 and angiotensin II AT1 receptors and signal transduction

Most frequently, the physiologic functions of the angiotensin II (Ang II) type 1 receptor (AT1R) and bradykinin B2 receptor (BKB2R) are antagonistic, particularly with respect to the regulation of vascular tone. Despite major differences in their physiologic actions, the receptors share sequence similarities. Both link to Galpha(i) and Galpha(q) and transduce very similar signal paths, not only those relating to the traditional G-protein associated second messengers, but also those involved in transactivation mechanisms involving receptor tyrosine kinases. With respect to these paths, some differences in signaling may be accounted for by cell type specificity. However, alternative signal cascades for these two receptors are becoming increasingly evident. One such is the recruitment of signaling molecules upon receptor translocation and internalization. The AT1R translocates into clathrin-coated pits and internalizes upon recruitment of beta-arrestin 2 which then recruits ASK1 and JNK3. The BKB2R translocates and internalizes mainly via caveolae. Another signaling divergence may be due to the direct activation of small G-proteins by both receptors. AT1R activates the RhoA, Rac1, Cdc42 while BKB2R couples only with Rac1 and Cdc42. Both receptors may serve as docking stations for intracellular proteins. One such example is the YIPP motif within the C-terminus of the ATIR which associates with the JAK/STAT pathway. Another potential alternative is the activation of tyrosine/serine kinase phosphatases by BK. This mechanism may directly oppose some of the protein tyrosine/ serine kinase paths activated by AT1R. These alternative mechanisms in sum are potentially responsible for the diversion in signal transduction between these two receptors. Regardless of the route of action, our results suggest that in Rat-1 fibroblasts stably transfected with BKB2R, BK slightly decreases connective tissue growth factor (CTGF) mRNA level while in ATIR transfected cells Ang II increases CTGF mRNA markedly. To determine whether mutant hybrids can be formed between these two receptors which encompass some of the function of the donor receptor but bind the ligand of the recipient receptor, a series of hybrids were formed with BKB2R the recipient and AT1R the donor receptor. Some of these hybrids show resistance to exchanges with the AT1R and form receptors which either do not bind (IC1 exchanges) or demonstrate poor function but normal internalization (proximal C-terminus exchanges). However, other hybrids have proven very functional. For example, the IC2, IC3 and distal C-terminus of the BKB2R IC face can be replaced simultaneously with the AT1R resulting in an hybrid which binds BK, continues to signal, is internalized and resensitized. Formation of this and other less extensive hybrids is discussed. Some of these hybrids possess the capacity to function as the AT1R as exemplified by their ability to upregulate CTGF expression as wild-type (WT) AT1R.     

Endogenous angiotensin II levels and the mechanism of action of angiotensin-converting enzyme inhibitors and angiotensin receptor type 1 antagonists

1. Modification of endogenous angiotensin II (AngII)-mediated processes by inhibitors of the angiotensin-converting enzyme (ACE) and antagonists of the angiotensin type 1 (AT(1) ) receptor is dependent upon both the levels of each agent in the plasma and tissues and on the concomitant changes in plasma and tissue AngII levels. 2. Both ACE inhibitors and AT(1) receptor antagonists increase renin secretion and angiotensin peptide formation in plasma and extrarenal tissues. Clinical doses of ACE inhibitors produce incomplete inhibition of ACE and the increased AngI levels act to restore AngII towards basal levels. Clinical doses of AT(1) receptor antagonists produce incomplete blockade of AT(1) receptors and the increased AngII levels in plasma and extrarenal tissues counteract (to an unknown degree) the effects of the antagonist. 3. The effects of ACE inhibitors and AT(1) receptor antagonists on AngII levels show tissue specificity. Angiotensin II-mediated processes in the kidney are most sensitive to inhibition by these agents. ACE inhibitors reduce renal AngII levels at doses much less than those required to reduce AngII levels in plasma and other tissues. Moreover, in contrast to increased AngII levels in plasma and extrarenal tissues, renal AngII levels do not increase in response to AT(1) receptor antagonists. The inhibition of AngII-mediated processes in the kidney may, therefore, play a primary role in mediating the effects of ACE inhibitors and AT(1) receptor antagonists on blood pressure and other aspects of cardiovascular function and structure. 4. Combination of an ACE inhibitor with an AT(1) receptor antagonist prevents the rise in plasma AngII levels that occurs with AT(1) receptor antagonism alone. This combination would, therefore, be predicted to produce more effective inhibition of endogenous AngII-mediated processes than either agent alone. We must await further studies to determine whether the combination of ACE inhibition and AT(1) receptor antagonism results in superior clinical outcomes.     

Intracellular angiotensin II elicits Ca2+ increases in A7r5 vascular smooth muscle cells

Recent studies show that angiotensin II can act within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane angiotensin II receptors. The signal transduction of intracellular angiotensin II is unclear. Therefore, we investigated the effects of intracellular angiotensin II in cells devoid of physiological responses to extracellular angiotensin II (A7r5 vascular smooth muscle cells). Intracellular delivery of angiotensin II was obtained by using liposomes or cell permeabilisation. Intracellular angiotensin II stimulated Ca2+ influx, as measured by 45Ca2+ uptake and single-cell fluorimetry. This effect was insensitive to extracellular or intracellular addition of losartan (angiotensin AT(1) receptor antagonist) or PD123319 ((s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate) (angiotensin AT2 receptor antagonist). Intracellular angiotensin II stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5,)P3) production and increased the size of the Ins(1,4,5,)P3 releasable 45Ca2+ pool in permeabilised cells, independent of losartan and PD123319. Small G-proteins did not participate in this process, as assessed by using GDPbetaS. Intracellular delivery of angiotensin I was unable to elicit any of the effects elicited by intracellular angiotensin II. We conclude from our intracellular angiotensin application experiments that angiotensin II modulates Ca2+ homeostasis even in the absence of extracellular actions. Pharmacological properties suggest the involvement of putative angiotensin non-AT1-/non-AT2 receptors.     

Characterization of the angiotensin II receptor expressed by the human hepatoma cell line, PLC-PRF-5.

Radioligand binding studies were undertaken to establish the expression of angiotensin II (AII) receptors on the human hepatoma cell line, PLC-PRF-5. Cell membranes were shown to express a large number of AII receptors with high and low affinity binding sites having Bmax values of 1269 +/- 365 and 4190 +/- 1055 fmol/mg protein and affinities (Kd) of 2.0 +/- 0.3 nM and 8.7 +/- 0.4 nM, respectively. In intact cells a single class of AII binding site was seen with an affinity (Kd) of 6.7 +/- 1 nM and a Bmax value of 315 +/- 32 fmol/mg. In both membranes and intact cells AII, AIII and the selective angiotensin AT1 receptor antagonist, DuP 753, all had a high affinity for the receptor (Ki values in the nanomolar range), but the selective angiotensin AT2 ligands, PD 123177 and p-aminophenylalanine6 AII, had low affinity (Ki values in the micromolar range). These results indicate that the PLC-PRF-5 cells express the angiotensin AT1 receptor subtype. This was further supported by the demonstration of the sensitivity of the receptor to dithiothreitol (DTT). Pretreatment of membranes with DTT reduced [3H]AII binding in a concentration-dependent manner with an IC50 of 4.2 +/- 0.9 mM. The coupling of the AT1 receptor to signal transduction pathways was investigated. In intact cells AII (100 nM) evoked an increase in intracellular calcium ([Ca2+]i). This increase in [Ca2+]i was unaffected by PD 123177 (100 microM) but was abolished by DuP 753 (100 microM). Furthermore, AII (100 nM) did not inhibit forskolin (0.1-10 microM) stimulated cyclic AMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular mechanisms of angiotensin II (AT1a) receptor endocytosis

1. Angiotensin II (AngII) initiates a variety of cellular responses through activation of type 1 (AT(1); with subtypes AT(1a) and AT(1b) ) and type 2 (AT(2) ) cell surface angiotensin receptors. Both AT(1) and AT(2) receptors couple to heterotrimeric guanyl nucleotide binding proteins (G-proteins) and generate intracellular signals following recognition of extracellular AngII, but only AT(1) is targeted for the rapid ligand-stimulated endocytosis (internalization) typical of many plasma membrane receptors. 2. AT(1) endocytosis proceeds through clathrin-coated pits and is independent of G-protein coupling which predicts that the AngII-AT(1) receptor complex attains a conformation necessary for interaction with the endocytotic machinery, but separate from receptor signalling activation. 3. The function of AT(1) endocytosis and the reason for the disparity between AT(1) and AT(2) endocytosis is not fully appreciated, but the latter probably reflects differences in the primary amino acid sequence of these two receptor types. 4. For many receptors that undergo internalization, it has been established that internalization motifs (2-6 amino acids, often incorporating crucial tyrosine and hydrophobic amino acids) within the cytoplasmic regions of the receptor mediate the selective recruitment of activated receptors into clathrin-coated pits and vesicles. 5. Mutagenesis studies on the AT(1a) receptor, aimed at identifying such motifs, reveal that sites within the third cytoplasmic loop and the cytoplasmic carboxyl terminal region are important for AngII-stimulated AT(1a) receptor endocytosis.     

The effect of angiotensin II and IV on ERK1/2 and CREB signalling in cultured rat astroglial cells

Angiotensin peptides produced by the brain renin-angiotensin system have established roles in cognition, but there is no mechanistic basis of angiotensin effects on memory. Astroglial cells present throughout the whole brain, synthesize all the components of the renin-angiotensin system and express angiotensin receptors; therefore our aim was to assess changes in intracellular signalling pathways related to memory formation, particularly the activation of CREB and ERK1/2 in astroglial cells grown in the presence of angiotensin peptides. Cultured rat astroglial cells were treated for 24 h with 10 μM angiotensin II and/or 10 μM angiotensin IV in the presence or absence of 100 μM losartan (AT1-receptor antagonist) or 100 μM PD123319 (AT2-receptor antagonist). Both angiotensin peptides alone were without effect on culture protein levels and cell viability and did not induce oxidative stress, but both peptides together slightly elevated cell growth rates and increased damaged, apoptotic cell numbers. This effect was most probably mediated by the AT1 receptor. Angiotensin II but not angiotensin IV increased intracellular calcium via activation of AT1 receptor. Angiotensin IV but not angiotensin II increased extracellular-regulated protein kinases 1 and 2 (ERK1/2) by 65% and T202, T204 phosphorylated ERK1/2 levels by 36%; this effect was blocked in part by both losartan and PD123319. Angiotensin II but not angiotensin IV increased cyclic AMP-responsive element binding protein (CREB) expression by almost 100% and elevated Ser 133-phosphorylated CREB levels by 56%. These effects were also inhibited in part by both losartan and PD123319. Our results indicate that CREB activation in cultured rat glial cells is mediated mostly by angiotensin II. Angiotensin IV appears to affect the ERK1/2 pathway.     

Differential signaling pathways in angiotensin II- and epidermal growth factor-stimulated hepatic C9 cells

Caveolin1 (Cav1) is an important component of the plasmamembrane microdomains, such as caveolae/lipid rafts, that are associated with angiotensin II type 1 (AT(1)) and epidermal growth factor (EGF) receptors in certain cell types. The interactions of Cav1 with other signaling molecules that mediate AT(1) receptor function were analyzed in angiotensin II (Ang II)- and EGF-stimulated hepatic C9 cells. This study demonstrated that cholesterol-rich domains mediate the actions of early upstream signaling molecules such as Src and intracellular Ca(2+) in cells stimulated by Ang II, but not by EGF, and that Cav1 has a scaffolding role in the process of mitogen-activated protein kinase activation. Furthermore, Cav1 phosphorylation by Ang II and EGF was regulated by intracellular Ca(2+) and Src, further indicating reciprocal interactions among Cav1, Src, and intracellular Ca(2+) through the AT(1) receptor. Phosphorylation of Cav1 and the EGF receptor by Ang II, but not of extracellular signal-regulated kinase 1/2, was dependent on intracellular Ca(2+). The phosphatidylinositol 3-kinase inhibitors, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and wortmannin, differentially modulated both Cav1 and EGF receptor activation by Ang II through intracellular Ca(2+). These findings further demonstrate the importance of Cav1 in conjunction with the receptor-mediated signaling pathways involved in cell proliferation and survival. It is clear that differential signaling pathways are operative in Ang II- and EGF-stimulated C9 cells and that cholesterol-enriched microdomains are essential components in cellular signaling processes that are dependent on specific agonists and/or cell types.     

Regulation of renal tubular sodium transport by angiotensin II and atrial natriuretic factor

1. The effects of angiotensin II (AngII) on water and electrolyte transport are biphasic and dose-dependent, such that low concentrations (10(-12) to 10(-9) mol/L) stimulate reabsorption and high concentrations (10(-7) to 10(-6) mol/L) inhibit reabsorption. Similar dose-response relationships have been obtained for luminal and peritubular addition of AngII. 2. The cellular responses to AngII are mediated via AT(1) receptors coupled via G-regulatory proteins to several possible signal transduction pathways. These include the inhibition of adenylyl cyclase, activation of phospholipases A(2), C or D and Ca(2+) release in response to inositol-1,4,5,-triphosphate or following Ca(2+) channel opening induced by the arachidonic acid metabolite 5,6,-epoxy-eicosatrienoic acid. In the brush border membrane, transduction of the AngII signal involves phospholipase A(2), but does not require second messengers. 3. Angiotensin II affects transepithelial sodium transport by modulation of Na(+) /H(+) exchange at the luminal membrane and Na(+)/HCO(3) cotransport, Na(+)/K(+)-ATPase activity and K(+) conductance at the basolateral membrane. 4. Atrial natriuretic factor (ANF) does not appear to affect proximal tubular sodium transport directly, but acts via specific receptors on the basolateral and brush border membranes to raise intracellular cGMP levels and inhibit AngII-stimulated transport. 5. It is concluded that there is a receptor-mediated action of ANF on proximal tubule reabsorption acting via elevation of cGMP to inhibit AngII-stimulated sodium transport. This effect is exerted by peptides delivered at both luminal and peritubular sides of the epithelium and provides a basis for the modulation by ANF of proximal glomerulotubular balance. The evidence reviewed supports the concept that in the proximal tubule, AngII and ANF act antagonistically in their roles as regulators of extracellular fluid volume.     

Induction of the angiotensin AT2 receptor subtype expression by differentiation of the neuroblastoma x glioma hybrid, NG-108-15.

In vitro differentiation of the mouse neuroblastoma-rat glioma hybrid cell line, NG-108-15, with dimethyl sulphoxide (1.5%) and low serum (0.5%), produced a marked increase in the number of angiotensin II receptors, from a level at the limit of sensitivity using labelled angiotensin II with a high specific activity ([125I]angiotensin II), in undifferentiated cells, to a Bmax of 1077 (1070-1268) fmol/mg in 5-day-differentiated cells. The affinity (Kd) of radiolabelled angiotensin II for the receptors in differentiated cells was 8.1 (7.5-10) nM. The recently available selective non-peptide antagonists, DuP 753 and PD 123177 and the peptide analogues of angiotensin II, CGP 42112A and p-aminophenylalanine6 angiotensin II, were used to characterize the angiotensin II receptors by competing for 125I-[Sar1-Ile8]angiotensin II binding to membranes prepared from undifferentiated and differentiated cells. The predominant angiotensin II receptor subtype expressed by undifferentiated cells was AT1 and after differentiation AT2. This change in receptor expression was evident 2 days after initiation of differentiation, was maximal at 4-5 days and was stable for at least 8 days. Administration of angiotensin II induced intracellular Ca2+ mobilization in both undifferentiated and differentiated cells. This was antagonised by the selective AT1 antagonist, DuP 753, indicating an action at the AT1 receptor subtype in both undifferentiated and differentiated cells. The selective AT2 antagonist, PD 123177 was without effect on the angiotensin II induced increase in intracellular Ca2+. This effect of DuP 753 on Ca2+ was specific for angiotensin II since the drug had no effect on bradykinin induced increases in intracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible involvement of calcium-calmodulin pathways in the positive chronotropic response to angiotensin II on the canine cardiac sympathetic ganglia

We investigated the ganglionic effects of angiotensin II (Ang II) and the signal transduction involved in the cardiac sympathetic ganglia by the direct administration of agents to the ganglia through the right subclavian artery and monitoring the heart rate as an indicator of the ganglionic function in pithed dogs. Ang II given i.a. caused increases in the heart rate, which was inhibited by the treatment with the AT1-receptor antagonist forasartan, but not by the AT2-receptor antagonist PD-123319. The stimulation by Ang II, but not by acetylcholine, was inhibited after treatment with an inhibitor of phospholipase C, U-73122; a cell-permeant modulator of the Ins(1,4,5)P3 receptors, 2-aminoethoxydiphenyl borate; an intracellular calcium and calcium-associated protein kinase inhibitor, HA-1077; calmodulin (CaM) inhibitor, W-7; Ca2+/CaM-dependent protein kinase II inhibitor, KN-93; a selective protein kinase C inhibitor, calphostin C; and Na+H+ exchange inhibitor, dimethylamiloride. These results suggest that Ang II stimulates the ganglionic transmission at postsynaptic sites via the activation of AT1 receptor coupled to either activation of phospholipase C, phosphoinositide hydrolysis and subsequent increase in intracellular Ca2+ and activation of protein kinase C and Ca2+/CaM kinase II, although this ganglionic stimulation seems to involve, at least in part, the protein kinases-dependent increase of amiloride-sensitive Na+ inflow.     

Immunosuppressive effect of angiotensin receptor blocker on stimulation of mice CTLs by angiotensin II

While angiotensin II, which is produced by the renin–angiotensin–aldosterone system, is considered to be the major regulator molecule that controls both the blood pressure and fluid system, there is an increasing body of evidence that this bioactive peptide and its receptor might also contribute to the immune system. However, there are few details known about the direct effect that angiotensin type I receptors (AT1R) have on the cytotoxic T cell (CTL). To clarify the relationship between angiotensin II and its CTL receptor, we used murine splenic and antigen-specific CTLs. Murine CTLs constantly expressed AT1R, with the activation of the AT1R expression strengthened by both anti-CD3 Ab and the use of an antigen-specific methodology. Moreover, the production of IFN-γ and TNF-α through CTL stimulation can be inhibited by the selective AT1R inhibitor, Losartan. In particular, the TNF-α production from activated CTL that had been magnified by angiotensin II, was nullified by the AT1R inhibitor. However, a cytotoxic assay indicated it did not have any effect on the cognate interaction of the CTLs. In addition, the antigen-specific CTL induction by immunization with the CTL antigenic peptide was reduced by angiotensin II type 1 receptor blocker (ARB) in vivo. These findings suggest that ARBs might have the ability to suppress excessive antigen-specific activation and induction of CTLs promoted by angiotensin II.