Low levels of AT1 receptor can make studying the growth-related signal transduction events mediated by this angiotensin II receptor in cardiac myocytes technically difficult. The purpose of the present study was to establish whether an adenovirus expression system could be used to increase the number of plasma membrane AT1 receptors in neonatal rat ventricular myocytes, thereby amplifying the signaling pathways activated by this receptor. Cardiac myocytes infected with adenovirus expressing the AT1 receptor exhibited increased ligand binding. The overexpressed receptor appeared to function like the endogenous receptor, in regard to agonist-induced internalization, as well as coupling to MAPK activation and protein tyrosine phosphorylation events. In addition, adenovirus-mediated overexpression of the AT1 receptor resulted in the amplification of angiotensin II intracellular signaling. In conclusion, adenovirus-mediated overexpression of angiotensin II receptors appears to be a useful strategy for studying the signal transduction events activated by this hormone in cardiac myocytes and for unraveling the molecular means by which this receptor type couples to a hypertrophic pattern of growth and gene expression.

Angiotensin II, the effector peptide of the renin-angiotensin system, regulates cellular growth in response to developmental, physiological, and pathological processes. The identification of renin-angiotensin system components and angiotensin II receptors in cardiac tissue suggests the existence of an autocrine/paracrine system that has effects independent of angiotensin II derived from the circulatory system. To be functional, a local renin-angiotensin system should produce sufficient amounts of the autocrine and/or paracrine factor to elicit biological responses, contain the final effector (angiotensin II receptor), and respond to humoral, neural, and/or mechanical stimuli. In this review, we discuss evidence for a functional cardiac renin-angiotensin system.

Changes in intracellular pH (pHi) can produce marked effects on cardiac function, and, therefore, it is important that the cell possess mechanisms by which pHi is regulated, especially after intracellular acidosis associated with myocardial ischemia. The Na+/H+ exchanger (NHE) and the Na+/HCO3− symport represent the 2 major pathways by which alkalinization occurs in cardiac cells. The NHE not only regulates pHi but also cell volume and intracellular signaling in response to a variety of stimuli.The NHE in mammalian myocardium1 has received considerable attention in the past decade, because it has been linked to cardiac growth and reperfusion injury.2 3 The NHE is activated by mechanical stretch in cultured cardiac myocytes and is thought to be a primary factor in influencing the anabolic state of the ventricular myocardium in response to pressure overload.4 5 Activation of the NHE occurs via phosphorylation of the cytoplasmic domain, which appears to be mediated by protein kinase C.6 Mechanical stretch of neonatal cardiac myocytes has also been associated with activation of second messengers, such as inositol triphosphate, protein kinase C, Raf-1 kinase, and mitogen-activated protein (MAP) kinase, all of which can contribute to reexpression of a number of fetal genes associated with cardiac hypertrophy, including β-myosin heavy chain, skeletal α-actin, and atrial natriuretic peptide.7 In studies using neonatal rat cardiac myocytes cultured on elastic membranes, stretch-mediated increases in MAP kinase activity and protein synthesis were attenuated by treatment of cells with the NHE antagonist Hoe 694,2 8 suggesting that the NHE has an important role in mediating growth in the pressure-overloaded myocardium.Deformation of the cardiac myocyte as it occurs in vivo, or in vitro under experimental or pathological conditions, presents a mechanical stress for cellular structures. This …

In cultured neonatal rat cardiac fibroblasts and CHO-K1 cells expressing angiotensin II (Ang II) type 1 receptors (AT 1 ) (T3CHO/AT 1A cell line), Ang II induced a delayed tyrosine phosphorylation of Stat3 (Signal Transducers and Activators of Transcription) with maximal activation at 2 h. This was in contrast to the rapid tyrosine phosphorylation (15–30 min) of Stat3 by the cytokine interleukin-6 (IL-6). Using T3CHO/AT 1A cells, we tested the hypothesis that the delayed tyrosine phosphorylation of Stat3 by Ang II resulted from the induction of an inhibitory pathway (0–30 min) prior to activation (1–2 h). In support of this hypothesis, we observed that a short treatment of cells with Ang II transiently inhibited the IL-6-induced Stat3 tyrosine phosphorylation. The inhibitory effect of Ang II could be attenuated by exposing the cells to a specific inhibitor of MAP kinase kinase 1, PD98059. Such modulatory cross-talk between Ang II and IL-6 may have relevance in pathophysiological conditions such as cardiac hypertrophy, and in acute phase in inflammatory responses.

We demonstrate a functional role for the 3'-untranslated region (3'-UTR) of the angiotensin II (Ang II) receptor subtype AT1A mRNA in Chinese hamster ovary (CHO-K1) cells by stably transfecting the coding region of the receptor gene with or without the 845 bp 3'-UTR. Two cell lines expressing similar levels of cell-surface receptors (with 3'-UTR, Bmax=571 fmol/mg protein; without 3'-UTR, Bmax=663 fmol/mg protein) were used in the present study. Both cell lines expressed high-affinity receptors (with 3'-UTR, Kd=0.83 nM; without 3'-UTR, Kd=0.82 nM), and binding studies with 125I-labelled Ang II in the presence of GTP[S] demonstrated that both coupled to heterotrimeric G-proteins. Despite these similarities, significant differences were observed for receptor-mediated cell signalling pathways. In cells without the 3'-UTR, Ang II stimulated an increase in cAMP accumulation (11-fold above control) and in cells with the 3'-UTR no stimulation was observed, which was consistent with previous observations in most endogenous Ang II receptor (AT1)-expressing cells. Activation of cAMP by Ang II in cells without the 3'-UTR correlated with an inhibition of DNA synthesis, determined by [3H]thymidine incorporation. Ang II-mediated responses were blocked by EXP3174, a selective non-peptide receptor antagonist. We also observed differences in the transient profiles of intracellular calcium between cells with and without the 3'-UTR in response to Ang II. In cells with the 3'-UTR, a sustained level of intracellular calcium was observed after Ang II stimulation, whereas cells without the 3'-UTR displayed a full return to basal level within 50 s of Ang II treatment. Even though the expressed exogenous gene is under the control of a constitutively expressing promoter (cytomegalovirus promoter), Northern-blot analysis revealed a considerably greater accumulation of AT1A mRNA in cells without the 3'-UTR compared with cells with the 3'-UTR. Analysis of the decay rate of the AT1A mRNA in cells with and without the 3'-UTR revealed that the normally unstable AT1A receptor mRNA became highly stable by removing its 3'-UTR, identifying a role for the 3'-UTR in mRNA destabilization. Interestingly, both cells express similar levels of receptors at the cell surface, suggesting that the 3'-UTR is also involved in the efficient translation and/or translocation of the receptor protein to the plasma membrane. We hypothesized that these 3'-UTR-mediated functions of the receptor are regulated by RNA-binding proteins. To identify possible RNA-binding proteins for the AT1A 3'-UTR, cellular extracts were prepared from parental CHO-K1 cells and 3'-UTR-binding assays, electrophoretic mobility-shift assays and UV crosslinking studies were performed. A major cellular protein of 55 kDa was identified, which specifically interacted with the 3'-UTR. Our data suggest that the 3'-UTR of the AT1A can control specific receptor functions, perhaps via selective recognition of the 3'-UTR by RNA-binding proteins.

Angiotensin II (Ang II), the effector peptide of the renin–angiotensin system (RAS), regulates volume and electrolyte homeostasis and is involved in cardiac and vascular cellular growth in humans and other species. This system, which has been conserved throughout evolution, plays an important role in cardiac and vascular pathology associated with hypertension, coronary heart disease, myocarditis and congestive heart failure. The traditional RAS is viewed as a system in which circulating Ang II is delivered to target organs and cells. However, in the past decade, a local RAS has been described in cardiac cells, providing evidence for autocrine and paracrine pathways by which biological actions of Ang II could be mediated. The critical actions of Ang II are mediated primarily through the AT1, G-protein (guanylyl nucleotide binding protein) coupled receptor. In addition to coupling to conventional G-protein signal transduction pathways, the AT1receptor was recently shown to increase the tyrosine phosphorylation of several intracellular substrates, including the STAT (Signal Transducers and Activators of Transcription) family of novel transcription factors, in rat cardiac fibroblasts, myocytes and vascular smooth muscle cells, and AT1receptor transfected CHO cells. It has been shown that Ang II stimulates the tyrosine phosphorylation and nuclear translocation of Stat1 (Stat 91) and Stat3 (Stat 92). Angiotensin II acting directly through the AT1receptor, induces the formation of a complex of STAT proteins termed SIF (sis-inducing factor) which binds the DNA sequence, SIE (sis-inducing element) present in the promotor element of many genes. This provides evidence for a direct role of Ang II in mediating inflammatory and remodeling responses through the JAK-STAT pathway. Thus, it is likely that the JAK-STAT pathway has an important role in Ang II-mediated effects on gene transcription, cardiac and vascular cellular growth/development, and inflammatory responses.

The spontaneously hypertensive rat (SHR) exhibits a transition from stable compensated left ventricular (LV) hypertrophy to heart failure (HF) at a mean age of 21 months that is characterized by a decrease in alpha-myosin heavy chain (alpha-MHC) gene expression and increases in the expression of the atrial natriuretic factor (ANF), pro-alpha1(III) collagen, and transforming growth factor beta1 (TGF-beta1) genes. We tested the hypotheses that angiotensin-converting enzyme inhibition (ACEI) in SHR would prevent and reverse HF-associated changes in gene expression when administered prior to and after the onset of HF, respectively. We also investigated the effect of ACEI on circulating and cardiac components of the renin-angiotensin system. ACEI (captopril 2 g/L in the drinking water) was initiated at 12, 18, and 21 months of age in SHR without HF and in SHR with HF. Results were compared with those of age-matched normotensive Wistar-Kyoto (WKY) rats, and to untreated SHR with and without evidence of HF. ACEI initiated prior to failure prevented the changes in alpha-MHC, ANF, pro-alpha1(III) collagen, and TGF-beta1 gene expression that are associated with the transition to HF. ACEI initiated after the onset of HF lowered levels of TGF-beta1 mRNA by 50% (P<.05) and elevated levels of alpha-MHC mRNA two- to threefold (P<.05). Circulating levels of renin and angiotensin I were elevated four- to sixfold by ACEI, but surprisingly, plasma levels of angiotensin II were not reduced. ACEI increased LV renin mRNA levels in WKY and SHR by two- to threefold but did not influence LV levels of angiotensinogen mRNA. The results suggest that the anti-HF benefits of ACEI in SHR may be mediated, at least in part, by effects on the expression of specific genes, including those encoding alpha-MHC, ANF, TGF-beta1, pro-alpha1(III) collagen, and renin-angiotensin system components.

1. Angiotensin II (AngII) initiates a variety of cellular responses through activation of type 1 (AT1; with subtypes AT1A and AT1B) and type 2 (AT2) cell surface angiotensin receptors. Both AT1 and AT2 receptors couple to heterotrimeric guanyl nucleotide binding proteins (G-proteins) and generate intracellular signals following recognition of extracellular AngII, but only AT1 is targeted for the rapid ligand-stimulated endocytosis (internalization) typical of many plasma membrane receptors. 2. AT1 endocytosis proceeds through clathrin-coated pits and is independent of G-protein coupling which predicts that the AngII-AT1 receptor complex attains a conformation necessary for interaction with the endocytotic machinery, but separate from receptor signalling activation. 3. The function of AT1 endocytosis and the reason for the disparity between AT1 and AT2 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 AT1A 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 AT1A receptor endocytosis.

Cardiac hypertrophy of diverse etiologies is associated with two remodeling events: an increase in cardiac muscle mass, and the abnormal accumulation of fibrillar collagen, which results in increased myocardial stiffness and eventual ventricular dysfunction. Clinical and animal studies have implicated angiotensin II (A II) as a growth promoter of both cardiac myocytes and fibroblasts during the cardiac remodeling that occurs with hypertension and myocardial infarction. The growth-promoting effects of A II occur, in part, independent of effects on hemodynamic load. Tissue culture studies have shown that cardiac myocytes and fibroblasts are targets for the actions of A II. In these cells. A II activates phospholipases C, D, and A2, leading in turn to the activation of multiple, conventional second-messenger pathways. By an undefined process. A II also increases the tyrosine phosphorylation of cytosolic proteins, and activates the STAT family of transcription factors, which may mediate an inflammatory or stress response. A II has been shown to affect gene expression of cultured cardiac myocytes and fibroblasts, induce either cellular hyperplasia or hypertrophy, and increase expression of other growth factors. Cardiac fibroblasts have been shown to respond to A II with increased expression of integrins and the extracellular matrix proteins, collagen and fibronectin. Recently, stretch of cardiac myocytes was shown to induce hypertrophy, through an autocrine release of A II. All of the aforementioned actions of A II are mediated by the AT1 receptor.

We reported recently that angiotensin II (AII) and phorbol 12-myristate 13-acetate (PMA) transiently inhibit interleukin 6 (IL-6)-stimulated tyrosine phosphorylation of signal transducers and activators of transcription 3 (Stat3) and subsequent formation of sis-inducing factor-A (SIF-A). However, the AII-mediated inhibition was independent of PMA-sensitive isoforms of protein kinase C (Bhat, G. J., Thekkumkara, T. J., Thomas, W. G., Conrad, K. M., and Baker, K. M. (1995) J. Biol. Chem. 270, 19059-19065). In this study, we demonstrate that the inhibition of IL-6-induced Stat3/SIF-A by AII is concentration-dependent and does not involve degradation of Stat3 protein. We hypothesized that the activation profile of the AII- and PMA-induced mitogen-activated protein (MAP) kinase cascade may be different from that of IL-6 and could contribute to the inhibitory effect; therefore, blocking the MAP kinase pathway at the level of MAPK kinase (MAPKK) would attenuate this inhibitory effect. AII and PMA rapidly induced high levels of MAP kinase activity (8-fold), which contrasted with the delayed and weak activation by IL-6 (1. 7-fold). Treatment of cells with PD98059, a specific inhibitor of MAPKK1, attenuated the inhibitory effects of AII and PMA on IL-6-induced Stat3 tyrosine phosphorylation and SIF-A formation. These data suggest that differences in magnitude and/or duration of activation of the MAP kinase cascade differentially affects the status of Stat3 tyrosine phosphorylation, and that MAPKK1 or a downstream intermediate is involved in the inhibition of IL-6-induced Stat3 by AII and PMA. Modulatory cross-talk between AII and IL-6 may have relevance in pathophysiological conditions such as cardiac hypertrophy and in acute phase and inflammatory responses.

We compared the ability of angiotensin II (Ang II) to induce hypertrophy of neonatal rat ventricular myocytes with that of endothelin-1. Over 72 hours, Ang II (1 mumol/L) increased the ratio of protein to DNA by less than 10%, whereas endothelin-1 (100 nmol/L) produced a 28% increase. The growth effects of either agonist occurred independently of chronotropic actions. Radioligand binding studies showed that myocytes have nearly 300-fold more receptors for endothelin-1 than Ang II, and type 1 and type 2 Ang II receptor subtypes (AT1 and AT2) are present in near equal proportions. Cotreatment with a 10-fold molar excess of AT2 antagonists (PD 123177 or CGP 42112) for 72 hours augmented the Ang II-induced increase in the protein-to-DNA ratio to levels nearly as high (23%) as those with endothelin-1 (28%). AT2 antagonists enhanced Ang II stimulation of protein synthesis, as indexed by [3H]leucine incorporation, whereas an AT1 antagonist blocked Ang II-induced incorporation. An AT2 antagonist also prevented Ang II-induced protein degradation. In conclusion, Ang II-induced myocyte growth is tempered because of low AT1 levels and an antigrowth effect of AT2. These findings have potential clinical significance in that regression of hypertension-induced cardiac hypertrophy by AT1 antagonists may be in part due to an unopposed antigrowth effect of Ang II mediated via AT2.