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Phosphorylation of serine and threonine residues in the carboxyl-terminal region of many G-protein-coupled receptors directs the rapid uncoupling from signal transduction pathways. In Chinese hamster ovary cells, we have stably expressed a truncated mutant of the angiotensin II (AT1A) receptor devoid of the carboxyl-terminal 45 amino acids, encompassing 13 serine/threonine residues. One clone, designated TL314 to indicate truncation after leucine 314, expressed a single class of angiotensin II receptors with a dissociation constant of 1.08 nM and a receptor density of 560 fmol/mg of protein (∼75,000 receptors/cell). A nonhydrolyzable analog of GTP accelerated the angiotensin II-induced dissociation of [125I]angiotensin II from TL314 plasma membranes 3.6-fold, indicating G-protein coupling. In TL314 cells, angiotensin II stimulated the release of intracellular calcium and the induction of mitogen-activated protein kinase activity, the levels of which were comparable with the full-length AT1A receptor. The AII-stimulated calcium response was rapidly desensitized in both full-length and truncated AT1A receptors. Interestingly, angiotensin II-induced endocytosis of the truncated receptor was almost completely inhibited, suggesting that a recognition motif within the carboxyl-terminal 45 amino acids of the AT1A receptor promotes sequestration. Thus, truncation of the AT1A receptor after leucine 314 inhibits agonist-induced internalization without affecting the capacity of the expressed protein to adopt the correct conformation necessary for high affinity binding of angiotensin II, coupling to G-proteins, and activation of signal transduction pathways. The rapid desensitization and refractoriness of the angiotensin II-induced calcium transient in the TL314 cell line, in which putative carboxyl-terminal phosphorylation sites are absent, suggests that the mechanism of AT1A receptor desensitization differs from that of other prototypical G-protein-coupled receptors.

Item does not contain fulltext The RAS (renin-angiotensin system) is one of the earliest and most extensively studied hormonal systems. The RAS is an atypical hormonal system in several ways. The major bioactive peptide of the system, AngII (angiotensin II), is neither synthesized in nor targets one specific organ. New research has identified additional peptides with important physiological and pathological roles. More peptides also mean newer enzymatic cascades that generate these peptides and more receptors that mediate their function. In addition, completely different roles of components that constitute the RAS have been uncovered, such as that for prorenin via the prorenin receptor. Complexity of the RAS is enhanced further by the presence of sub-systems in tissues, which act in an autocrine/paracrine manner independent of the endocrine system. The RAS seems relevant at the cellular level, wherein individual cells have a complete system, termed the intracellular RAS. Thus, from cells to tissues to the entire organism, the RAS exhibits continuity while maintaining independent control at different levels. The intracellular RAS is a relatively new concept for the RAS. The present review provides a synopsis of the literature on this system in different tissues. 01 september 2012

Angiotensin (ANG) II has been previously shown to stimulate proliferation of neonatal rat cardiac fibroblasts via AT1 receptors. Here we conducted studies to assess involvement in this process of two second messengers linked to AT1 receptors, protein kinase C (PKC) and Ca2+. Several findings argue against a dominant role for PKC in ANG II-induced mitogenesis: 1) [Sar1]ANG II, which produced a modest, transient increase in PKC activity, was equally effective in inducing thymidine incorporation into DNA in PKC-depleted cells, whereas the effect of platelet-derived growth factor (PDGF)-BB on thymidine incorporation was reduced to the level observed with [Sar1]ANG II; 2) phorbol 12-myristate 13-acetate (PMA), a potent PKC stimulator, was ineffective in stimulating thymidine incorporation; and 3) PKC downregulation or the highly specific PKC inhibitor, compound 3, eliminated PMA-induced mitogen-activated protein (MAP) kinase activity but did not affect comparable increases induced by [Sar1]ANG II or PDGF-BB. Increased intracellular Ca2+ may be sufficient to account for [Sar1]ANG II-induced MAP kinase activity because ionomycin also increased MAP kinase activity and chelation of intracellular Ca2+ eliminated [Sar1]ANG II-induced activity in PKC-depleted fibroblasts. However, Ca2+ chelation did not prevent [Sar1]ANG II-induced MAP kinase activity in non-PKC-depleted fibroblasts. Thus ANG II can activate MAP kinase in cardiac fibroblasts by either Ca(2+)- or PKC-dependent pathways, and whereas the full effect of PDGF-BB on thymidine incorporation and cell proliferation requires a phorbol ester-sensitive PKC, the hyperplastic growth effect of ANG II does not.

Angiotensin II has been demonstrated to act as a growth factor in rat cardiac fibroblasts. However, the signaling events that lead to fibroblast cell growth in response to angiotensin II remain to be elucidated. This study was designed to determine whether angiotensin II stimulated tyrosine phosphorylation of proteins in cardiac fibroblasts. Immunoblot analysis demonstrated rapid tyrosine phosphorylation of distinct substrates of 125, 95, 46-60, and 44 kDa in response to 10 nM angiotensin II. Tyrosine phosphorylation was maximal at 5 min and persisted for at least 180 min. Additional tyrosine-phosphorylated proteins of 185, 145, and 85 kDa were detected in response to 10 ng/ml platelet-derived growth factor BB. A cluster of 75-80-kDa proteins were phosphorylated in response to angiotensin II, phorbol ester, and platelet-derived growth factor. Angiotensin II-induced tyrosine phosphorylation was unaffected by phorbol ester-sensitive protein kinase C down-regulation and could be partially blocked by pertussis toxin pretreatment. Angiotensin II stimulation resulted in increased cytosolic tyrosine kinase activity which was recovered by immunoprecipitation. Immunoblot analysis demonstrated tyrosine phosphorylation of p44MAPK, and, in addition, we demonstrated for the first time tyrosine phosphorylation of p125FAK, p46SHC, and p56SHC in response to angiotensin II. The finding that angiotensin II and platelet-derived growth factor stimulated tyrosine phosphorylation of p46SHC and p56SHC suggested that this protein may serve as a common tyrosine kinase substrate in the mitogenic signaling cascade induced by G-protein-coupled receptors and growth factors and is consistent with the hypothesis that angiotensin II-induced tyrosine phosphorylation is involved in mitogenic signaling pathways in neonatal rat cardiac fibroblasts.

The renin-angiotensin system (RAS) has mainly been categorized as a circulating and a local tissue RAS. A new component of the local system, known as the intracellular RAS, has recently been described. The intracellular RAS is defined as synthesis and action of ANG II intracellularly. This RAS appears to differ from the circulating and the local RAS, in terms of components and the mechanism of action. These differences may alter treatment strategies that target the RAS in several pathological conditions. Recent work from our laboratory has demonstrated significant upregulation of the cardiac, intracellular RAS in diabetes, which is associated with cardiac dysfunction. Here, we have reviewed evidence supporting an intracellular RAS in different cell types, ANG II's actions in cardiac cells, and its mechanism of action, focusing on the intracellular cardiac RAS in diabetes. We have discussed the significance of an intracellular RAS in cardiac pathophysiology and implications for potential therapies.

Angiotensin II has been reported to be a hormonal stimulus of cardiac growth, a response that may involve myocyte hypertrophy as well as growth of nonmyocytes. This study was designed to determine whether neonatal rat cardiac fibroblasts have an angiotensin II receptor that is coupled with hypertrophic and/or proliferative growth. Competitive radioligand binding studies showed that cardiac fibroblasts have a single class of high-affinity (IC50, 1.0 nM) angiotensin II binding sites (Bmax, 778 fmol/mg protein) that are sensitive to the competitive nonpeptide AT1 receptor antagonist losartan (IC50, 13 nM). Other angiotensin peptides competed for [125I]angiotensin II binding in the following rank order: angiotensin II > angiotensin III > angiotensin I > > [des-Asp1-des-Arg2]angiotensin II. A nonhydrolyzable analogue of guanosine triphosphate increased the dissociation rate of bound [125I]angiotensin II and decreased hormone binding to the receptor at equilibrium. The angiotensin II receptor was coupled with increases in intracellular calcium. Incorporation of precursors into protein, DNA, and RNA in response to angiotensin II was determined. In serum-deprived cultures, a 24-hour exposure to 1 microM [Sar1]angiotensin II increased rates of phenylalanine, thymidine, and uridine incorporation by 58%, 103%, and 118%, respectively. These increases were blocked by the noncompetitive AT1 receptor antagonist EXP3174. After 48 hours, [Sar1]angiotensin II increased total protein and DNA of cardiac fibroblasts by 23% and 15%, respectively, with no change in the protein/DNA ratio. [Sar1]Angiotensin II increased cell number by 138% after a 24-hour exposure, without affecting cell area. In summary, cardiac fibroblasts have G protein-linked AT1 receptors that are coupled with proliferative growth. These results suggest that angiotensin II-induced cardiac hypertrophy is, in part, secondary to stimulated increases in nonmyocyte cellular growth.

Although substantial evidence of a cardiac RAS has been obtained in the past decade, a number of important questions remain unanswered. These include identification and localization of the cell types responsible for production of the system's components as well as the regulation of synthesis, storage, and secretion pathways for each component. Future studies, which will utilize tools of molecular biology that have become recently available (for example, transgenic animal models), renin inhibitors, angiotensin receptor antagonists, and bradykinin antagonists, will help to elucidate specific roles of the cardiac RAS in normal and failing hearts.

Background / Aim: The upregulation of the renin-angiotensin system (RAS) represents a major pathological mechanism in diabetes. We have previously reported that hyperglycemia preferentially increases intracellular generation of angiotensin (Ang) II. However, circulating RAS is down regulated in diabetes implying the accelerated intracellular RAS mechanism as a major contributor to diabetic cardiomyopathy. In this study, we determined expression of RAS components and their effect on cardiac cells to give us a venue to intervene. Methods: Diabetes was induced in Sprague-Dawley rats and C57/BL6J mice with injection of streptozotocin for 5 days and verified by sustained blood glucose levels >15 mmol/L. Control mice received buffered saline alone. One week after diabetes induction the mice hearts were removed and perfused by the Langendorff method. Myocytes were isolated with enzymatic dispersion and centrifugation. RAS proteins were determined by real-time PCR and Western analysis. These included angiotensinogen, renin, angiotensin converting enzyme, AT1, AT2, and ACE 2. Angiotensin II was purified by reverse-phase chromatography and quantified by competitive ELISA. Results: Among cells obtained from diabetic hearts, expression of AGT (3.5+/− 0.8 fold), renin (2.4 +/− 0.4), and AT1 (2.6+/− 0.3) was significantly increased compared to cells from control hearts (p

The renin-angiotensin system has a varied role in the regulation of cardiac function, ranging from early receptor-mediated effects such as second messenger generation, to more delayed responses such as protein synthesis and cell growth. Clinically, the importance of the RAS in cardiovascular disease is becoming increasingly evident with the use of ACE inhibitors in treating various pathological processes. With evidence for the existence of a local RAS in the heart, the molecular and biochemical regulation of this system requires investigation. Much additional work needs to be directed toward elucidating the mechanisms by which the AII-receptor couples to cardiac growth, how the local RAS is regulated, and the nature of controls that modulate cardiac production and actions of this peptide. Increased understanding of the mechanisms by which AII actions are affected in cardiac tissue will likely lead to enhanced therapeutic modalities for the treatment of pathological cardiovascular conditions in which the RAS plays an integral role.

Angiotensin II (ANG II) is a stimulus for positive chronotropic and inotropic effects, protein synthesis, and hypertrophic growth in cardiac tissue. These short- and long-term effects of ANG II are mediated through specific plasma membrane receptors. Indirect evidence suggests that ANG II synthesized in the myocardium may be important in regulating cardiac function. The cell types in the myocardium that produce components of the renin-angiotensin system have not been determined. In this study, we evaluated whether cultured cardiomyocytes and fibroblasts obtained from ventricles of neonatal rat hearts were capable of synthesizing ANG I and II. Both cardiomyocytes and fibroblasts were found to have immunofluorescent staining for ANG I, ANG II, and angiotensin-converting enzyme (ACE). The amounts of ANG I and II in cell extracts and conditioned media obtained from cardiomyocytes and fibroblasts were quantified by radioimmunoassay. The amounts of ANG I and II detected in cardiomyocyte cultures (1.48 x 10(6) cells/dish) were 32.2 +/- 16.2 (n = 4) and 6.2 +/- 2.9 (n = 4) ng/10(6) cells, respectively. The amounts of ANG I and II detected in the media conditioned by a 48-h exposure to cardiomyocytes were 5.2 +/- 1.2 (n = 3) and 2.1 +/- 1.2 (n = 3) ng/10(6) cells, respectively. The amounts of ANG I and II detected in fibroblast cultures (5.38 x 10(6) cells/dish) were 34.8 +/- 4.9 (n = 4) and 8.0 +/- 3.5 (n = 4) ng/10(6) cells, respectively. The amounts of ANG I and II obtained from media conditioned by a 48-h exposure to fibroblasts were 4.7 +/- 0.6 (n = 4) and 3.3 +/- 2.1 (n = 4) ng/10(6) cells, respectively. The identity of the radioimmunoassayable materials as ANG I and II peptides was confirmed in cardiomyocytes using an in vitro bioassay based on displacement of 125I-ANG II from receptor binding sites in cardiac membranes prepared from neonatal pig heart. Identification of ANG I and II and ACE in vitro in cultures of cardiac myocytes and fibroblasts supports the hypothesis that there is an intracardiac renin-angiotensin system that produces these peptides.

Angiotensin II (AII) has been reported to have direct hypertrophic actions in mammalian and avian myocardium. In this study we determined whether AII had receptor-mediated effects on stimulating cardiac hypertrophy independent of mechanical stimuli (increased cardiac afterload) in adult rats. Angiotensin II was infused into Sprague-Dawley rats for 7 and 14 days. Following this infusion, left ventricular mass indexed to body weight (LV/BW) increased 18.6 and 17.3%, respectively, compared with control (saline infused) rats. Administration of the nonpeptide AII receptor antagonist Dup 753 prevented the increase in left ventricular hypertrophy. Blockade of converting enzyme with enalapril maleate and treatment with a vasodilator had no effect on the AII-induced hypertrophy. In this animal model, the cardiac hypertrophy appeared to be independent of cardiac afterload, because normalization of blood pressure with hydralazine did not prevent the AII-induced hypertrophy. These in vivo studies indicate that AII-induced cardiac hypertrophy is mediated through AT1 angiotensin receptors.

Recently, several novel aspects of the renin-angiotensin system (RAS) were described, which potentially may change the therapeutic strategy to treat cardiovascular disease, in addition to enhancing understanding of this system’s mechanism of action. Most notably, identification of a functional intracellular RAS may address several unanswered questions regarding a direct role of angiotensin (Ang) II in cardiac remodeling and incomplete efficacy of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers or superiority of a renin inhibitor in cardiovascular disorders. We describe the physiology of the intracellular RAS, potential pathologic roles of intracellular Ang II, and the relevance of the intracellular system in view of recent clinical trials involving various RAS inhibitors.

Involvement of the renin–angiotensin system (RAS) in human pathophysiology has expanded to include several diseases beyond a traditional role in salt and water homeostasis. In diabetes, there is significant overactivity of the RAS, which is inhibited by treatment with RAS blockers, thus decreasing diabetic complications. Activation of the RAS in diabetes includes several unique aspects, such as elevation of circulating prorenin levels and angiotensin (Ang) II-independent effects, mediated through interaction of pro(renin), with the pro(renin) receptor. Ang II-independent RAS actions suggest that efficacy of angiotensin receptor blockers (ARBs) and ACE inhibitors would have limitations in the treatment of diabetic patients. Recent meta-analyses of clinical trials have suggested that currently used RAS blockers may not provide additional benefits in diabetics compared to non-diabetics. We recently reported another novel aspect of the RAS, the intracellular system, which is dramatically activated in hyperglycemic conditions. In cardiac myocytes and fibroblasts, we demonstrated the presence of RAS components and synthesis of Ang II intracellularly. Hyperglycemia selectively upregulated the intracellular system in cardiac myocytes, vascular smooth muscle cells (VSMC), and renal mesangial cells where Ang II synthesis was largely catalyzed by chymase, not ACE. We also demonstrated elevation of intracellular Ang II (iAng II) levels in diabetic rat hearts, which resulted in increased cardiac myocyte apoptosis, oxidative stress, and cardiac fibrosis, suggesting a significant role of iAng II in diabetic cardiomyopathy. Others and we have previously reported that iAng II elicits multiple biological effects, some of which are not blocked by ARBs. Using Chinese hamster ovary (CHO) cells that do not express AT1 receptor, we confirmed that the latter are not required for intracellular actions of Ang II. The AT1-independent effects of iAng II are likely mediated by novel interactions between Ang II and intracellular proteins. The mechanism of RAS activation and intracellular accumulation of components by cardiac myocytes in high glucose (HG) conditions is not known. There is a possibility that an increased influx of glucose into the hexosamine biosynthesis pathway (HBP) and resultant O-glycosylation of proteins/transcription factors is responsible for the activation of the RAS and intracellular synthesis of Ang II.

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Cardiac hypertrophy usually follows an increase in work load that is imposed on the heart. Usually, greater work is due to increased afterload in patients with hypertension or after myocardial infarction in whicfh the remaining muscle must assume the work of the segment that was lost as a result of myocardial cell death and scar formation. In some instances, increased pressure or volume work of the left ventricle is associated with increased release of neurotransmitters or plasma concentrations of hormones that may also have direct effects on cardiac myocyte growth, including activation of the sympathetic nervous system, pheochromocytoma, hyperthyroidism, and high renin hypertension

The renin-angiotensin system, traditionally viewed as a circulatory system, has significantly expanded in the last two decades to include independently regulated local systems in several tissues, newly identified active products of angiotensin II, and new receptors and functions of renin-angiotensin system components. In spite of our increased understanding of the renin-angiotensin system, a role of angiotensin II in cardiac hypertrophy, through direct effects on cardiovascular tissue, is still being debated. Here, we address the cardiovascular effects of angiotensin II and the role an intracellular renin-angiotensin system might play.Recent studies have shown that cardiac myocytes, fibroblasts and vascular smooth muscle cells synthesize angiotensin II intracellularly. Some conditions, such as high glucose, selectively increase intracellular generation and translocation of angiotensin II to the nucleus. Intracellular angiotensin II regulates the expression of angiotensinogen and renin, generating a feedback loop. The first reaction of intracellular angiotensin II synthesis is catalyzed by renin or cathepsin D, depending on the cell type, and chymase, not angiotensin-converting enzyme, catalyzes the second step.These studies suggest that the intracellular renin-angiotensin system is an important component of the local system. Alternative mechanisms of angiotensin II synthesis and action suggest a need for novel therapeutic agents to block the intracellular renin-angiotensin system.

The octapeptide [Ile5]angiotensin II (ANG II), which is the principal circulating hormone of the renin-angiotensin system, could modulate or mediate cardiac hypertrophy via indirect effects, through increases in total peripheral vascular resistance, or by direct effects on cardiac cells, which result in increased protein synthesis and cell growth. In this study we determined whether ANG II stimulated protein synthesis and cell growth in cultures of embryonic chick myocytes. After 3 h of exposure to ANG II, there were significant increases in total cellular protein at 120, 144, and 168 h and in the relative rate of protein synthesis at 120 and 144 h. There was a significant increase in the fractional rate of protein synthesis of 32.2% (0.0119 +/- 0.0008 h-1 for ANG II stimulated and 0.0090 +/- 0.0003 h-1 for control). The stimulatory effects of ANG II on protein synthesis and cell growth were inhibited by the ANG II antagonist [Sar1,Ile8]ANG II and the hexapeptide ANG II-(3-8). ANG II significantly increased total RNA levels in myocytes, at 12 h after exposure to the peptide. The stimulatory effect of ANG II (32%) on total cellular protein was slightly greater than that seen with norepinephrine (20%) in contrast to the greater stimulatory effect seen with phorbol 12,13-dibutyrate (47%). ANG II and [Sar1,Ile8]ANG II each stimulated increases in cytosolic-free Ca2+, whereas ANG II-(3-8) did not. Growth-related effects of changes in the chronotropic state of the myocytes were excluded, in that, ANG II-stimulated increases in protein synthesis and cellular protein were not inhibited by potassium chloride depolarization of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac hypertrophy is a process that occurs in response to various mechanical or hormonal stimuli. Stimulation of the renin-angiotensin system is involved in the process of cardiac hypertrophy through mechanisms related to increased peripheral vascular resistance and increased cardiac afterload. In this study we determined whether [Sar1]angiotensin II (ANG II) directly stimulated protein synthesis and cell growth in embryonic chick myocytes in cell culture. Eighteen-day-old embryonic chick myocytes in subconfluent cell culture, incubated in a chemically defined serum-free media, showed a significant increase in total protein content, 18.5, 26.2, and 22.2%, respectively, when exposed to [Sar1]ANG II (1 microM/day) for 5, 7, and 9 days, respectively. The increase in total protein resulted in part from an increase in the fractional protein synthesis rate of 21.7, 16.5, and 14.9% at 5, 7, and 9 days, respectively. Total DNA and RNA levels did not change significantly following a 4-day exposure to [Sar1]ANG II in subconfluent culture. The relative rate of protein synthesis, determined by pulse labeling for 3 h with [3H]phenylalanine, showed increases of 23.4, 22.9, and 17.8% over control after 4, 5, and 6 days of exposure to [Sar1]ANG II. The incorporation of [3H]phenylalanine was blocked by the specific ANG II-receptor antagonist [Sar1,Ile8]ANG II. The data demonstrate a receptor-mediated increase in the rate of protein synthesis in cultured chick myocytes in response to [Sar1]ANG II, with a resultant increase in total cellular protein. This angiotensin peptide appears to directly stimulate protein synthesis in cultured embryonic chick myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiomyocyte apoptosis has an important role in the transition from compensatory cardiac remodeling to heart failure. All-trans retinoic acid (RA), a bioactive vitamin A derivative, prevents stretch- and angiotensin II (Ang II)-induced cardiac hypertrophy. However, the anti-apoptotic potential of RA in the heart remains unexplored. Here, we demonstrate that stretch- and Ang II-induced apoptosis is prevented by RA in neonatal cardiomyocytes. RA improved mitochondrial function by inhibiting the stretch- and Ang II-induced reduction in mitochondrial membrane potential, cytochrome c release and by increasing the Bcl2/Bax ratio. RA inhibited stretch- and Ang II-induced intracellular reactive oxygen species (ROS) generation and upregulated the SOD2 level. Hydrogen peroxide-induced increases in the number of TUNEL-positive cells and percentage of Annexin V positive cells, were dose-dependently inhibited by RA. The thiol antioxidant, N-acetyl cysteine (NAC), completely inhibited stretch- and Ang II-induced apoptosis. Using diazoxide (mitochondrial ATP-sensitive K(+) channel opener) and SDS (NADPH oxidase activator), we confirmed that RA suppressed both mitochondrial- and NADPH oxidase-derived ROS. We also observed that both RAR and RXR were involved in preventing Ang II- and stretch-induced ROS production and apoptosis, by using selective retinoid receptor agonists and antagonists. Our data provide the first evidence that RA prevents Ang II and stretch induced apoptosis, by inhibiting ROS generation and increasing the anti-oxidant defense system, suggesting that RA-mediated signaling may provide a new therapeutic target for the prevention of the cardiac remodeling process.

Retinoic acid (RA), the active derivative of vitamin A, by acting through retinoid receptors, is involved in signal transduction pathways regulating embryonic development, tissue homeostasis, and cellular differentiation and proliferation. RA is important for the development of the heart. The requirement of RA during early cardiovascular morphogenesis has been studied in targeted gene deletion of retinoic acid receptors and in the vitamin A-deficient avian embryo. The teratogenic effects of high doses of RA on cardiovascular morphogenesis have also been demonstrated in different animal models. Specific cardiovascular targets of retinoid action include effects on the specification of cardiovascular tissues during early development, anteroposterior patterning of the early heart, left/right decisions and cardiac situs, endocardial cushion formation, and in particular, the neural crest. In the postdevelopment period, RA has antigrowth activity in fully differentiated neonatal cardiomyocytes and cardiac fibroblasts. Recent studies have shown that RA has an important role in the cardiac remodeling process in rats with hypertension and following myocardial infarction. This chapter will focus on the role of RA in regulating cardiomyocyte growth and differentiation during embryonic and the postdevelopment period.

More than a century after its discovery, the physiological implications of the renin–angiotensin system (RAS) continue to expand, with the identification of new components, functions and subsystems. These advancements have led to better management and understanding of a broad range of cardiovascular and metabolic disorders. The RAS has traditionally been viewed as a circulatory system, involved in the short-term regulation of volume and blood pressure homeostasis. Recently, local RASs have been described as regulators of chronic tissue effects. Most recently, studies have provided evidence of a complete, functional RAS within cells, described as an ‘intracrine' or intracellular system. A more comprehensive understanding of the intracellular RAS provides for new strategies in system regulation and a more efficacious approach to the management of RAS-related diseases.

Angiotensin II (Ang II), a peptide hormone, is the primary product of the renin-angiotensin system (RAS). The main effects of Ang II are related to the control of cardiovascular, renal and adrenal functions, as related to fluid and electrolyte balance, and arterial pressure. In addition, the octapeptide Ang II also affects cell growth, apoptosis, fibrosis, inflammation, and coagulation. The classical RAS involves the biosynthesis of the enzyme renin in the juxtaglomerular cells of the kidney. Active renin cleaves angiotensinogen (Ao), which is synthesized in the liver to form the decapeptide angiotensin I (Ang I). Angiotensin I is acted upon by angiotensin-converting enzyme (ACE), a primarily membrane bound glycoprotein, which hydrolyzes the Ang I into the biologically active Ang II. The majority of the actions of Ang II are attributed to the interaction of the peptide with the Ang II, type 1 plasma membrane (AT1) receptor. The AT1 is a seven-transmembrane spanning, heterotrimeric G protein-coupled receptor (GPCR) that activates multiple signal transduction pathways. An AT2 receptor, which in large part couples to effector pathways that antagonize the AT1 effects, is also present in the cardiovascular tissue. The AT2 receptor function will be discussed in more detail in Chapter 5. In addition to the classical, circulating RAS, locally derived RASs have been described in many studies on organs and tissues, including the heart and vasculature. Clinical, animal, and cell culture studies support the concept that these local RASs can act in an autocrine, paracrine, and/or intracrine manner.

Retinoic acid (RA), the active derivative of vitamin A, by acting through retinoid receptors, is involved in signal transduction pathways regulating embryonic development, tissue homeostasis, and cellular differentiation and proliferation. RA is important for the development of the heart. The requirement of RA during early cardiovascular morphogenesis has been studied in targeted gene deletion of retinoic acid receptors and in the vitamin A-deficient avian embryo. The teratogenic effects of high doses of RA on cardiovascular morphogenesis have also been demonstrated in different animal models. Specific cardiovascular targets of retinoid action include effects on the specification of cardiovascular tissues during early development, anteroposterior patterning of the early heart, left/right decisions and cardiac situs, endocardial cushion formation, and in particular, the neural crest. In the postdevelopment period, RA has antigrowth activity in fully differentiated neonatal cardiomyocytes and cardiac fibroblasts. Recent studies have shown that RA has an important role in the cardiac remodeling process in rats with hypertension and following myocardial infarction. This chapter will focus on the role of RA in regulating cardiomyocyte growth and differentiation during embryonic and the postdevelopment period.

We recently reported intracrine effects of angiotensin II (ANG II) on cardiac myocyte growth and hypertrophy that were not inhibited by the ANG II type 1 receptor (AT1) antagonist, losartan. To further determine the role of AT1 in intracrine effects, we studied the effect of intracellular ANG II (iANG II) on cell proliferation in native Chinese hamster ovary (CHO) cells and those stably transfected with AT1 receptor (CHO-AT1). CHO-AT1, but not CHO cells, showed enhanced proliferation following exposure to extracellular ANG II (eANG II). However, when transiently transfected with an iANG II expression vector, both cell types showed significantly enhanced proliferation, compared with those transfected with a scrambled peptide. Losartan blocked eANG II-induced cell proliferation, but not that induced by iANG II. To further confirm these findings, CHO and CHO-AT1 cells were stably transfected for iANG II expression (CHO-iA and CHO-AT1-iA, respectively). Cells grown in serum-free medium were counted every 24 h, up to 72 h. CHO-iA and CHO-AT1-iA cells showed a steeper growth curve compared with CHO and CHO-AT1, respectively. These observations were confirmed by Wst-1 assay. The AT1 receptor antagonists losartan, valsartan, telmisartan, and candesartan did not attenuate the faster growth rate of CHO-iA and CHO-AT1-iA cells. eANG II showed an additional growth effect in CHO-AT1-iA cells, which could be selectively blocked by losartan. These data demonstrate that intracrine ANG II can act independent of AT1 receptors and suggest novel intracellular mechanisms of action for ANG II.

All-trans retinoic acid (RA) has been implicated in mediation of cardiac growth inhibition in neonatal cardiomyocytes. However, the associated signaling mechanisms remain unclear. Utilizing neonatal cardiomyocytes, we demonstrated that RA suppressed the hypertrophic features induced by cyclic stretch or angiotensin II (Ang II). Cyclic stretch- or Ang II-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAP kinase) was dose- and time-dependently inhibited by RA. Significant inhibition was observed by 5 microm RA, from 8 to 24 h of pretreatment. This inhibitory effect was not mediated at the level of mitogen-activated protein kinase kinases (MKKs), because RA had no effect on stretch- or Ang II-induced phosphorylation of MEK1/2, MKK4, and MKK3/6. However, the phosphatase inhibitor vanadate reversed the inhibitory effect of RA on MAP kinases and protein synthesis. RA up-regulated the expression level of MAP kinase phosphatase-1 (MKP-1) and MKP-2, and the time course was correlated with the inhibitory effect of RA on activation of MAP kinases. Overexpression of wild-type MKP-1 inhibited the phosphorylation of JNK and p38 in cardiomyocytes. These data indicated that MKPs were involved in the inhibitory effect of RA on MAP kinases. Using specific RAR and RXR antagonists, we demonstrated that both RARs and RXRs were involved in regulating stretch- or Ang II-induced activation of MAP kinases. Our findings provide the first evidence that the anti-hypertrophic effect of RA is mediated by up-regulation of MKPs and inhibition of MAP kinase signaling pathways.

Gastrin and its precursors promote proliferation in different gastrointestinal cells. Since mature, amidated gastrin (G-17) can induce cyclin D1, we determined whether G-17-mediated induction of cyclin D1 transcription involved Wnt signaling and CRE-binding protein (CREB) pathways. Our studies indicate that G-17 induces protein, mRNA expression and transcription of the G(1)-specific marker cyclin D1, in the gastric adenocarcinoma cell line AGSE (expressing the gastrin/cholecystokinin B receptor). This was associated with an increase in steady-state levels of total and nonphospho beta-catenin and its nuclear translocation, indicating the activation of the Wnt-signaling pathway. In addition, G-17-mediated increase in cyclin D1 transcription was significantly attenuated by axin or dominant-negative (dn) T-cell factor 4(TCF4), suggesting crosstalk of G-17 with the Wnt-signaling pathway. Mutational analysis indicated that this effect was mediated through the cyclic AMP response element (CRE) (predominantly) and the TCF sites in the cyclin D1 promoter, which was also inhibited by dnCREB. Furthermore, G-17 stimulation resulted in increased CRE-responsive reporter activity and CREB phosphorylation, indicating an activation of CREB. Chromatin immunoprecipitation studies revealed a G-17-mediated increase in the interaction of beta-catenin with cyclin D1 CRE, which was attenuated by dnTCF4 and dnCREB. These results indicate that G-17 induces cyclin D1 transcription, via the activation of beta-catenin and CREB pathways.

Beta-arrestins play a role in AT1 endocytosis by binding the cytoplasmic, C-terminus region T332-S338, the major site of angiotensin II (Ang II)-induced phosphorylation. However, the processes responsible for recruiting beta-arrestin to the activated receptor are poorly defined. In this study, we used CHO-K1 and HEK 293 cells expressing wild-type or mutant AT1 to investigate two possibilities: activated AT1 induces global relocation of beta-arrestins to the plasma membrane or the phosphorylated C-terminus acts as bait to attract beta-arrestins. Results obtained using high osmolarity and dominant-negative beta-arrestin confirmed that internalization of AT1 in both CHO-K1 and HEK 293 cells is predominately via clathrin-mediated endocytosis involving beta-arrestin, and substitution of T332, S335, T336 and S338 with alanine to preclude phosphorylation markedly attenuated AT1 internalization. Confocal microscopy revealed that wild-type AT1 induced a time-dependent translocation of GFP-tagged beta-arrestins 1 and 2 to the cell surface. In contrast, the TSTS/A mutant did not traffic beta-arrestin 1 at all, and only trafficked beta-arrestin 2 weakly. Results of rescue-type experiments were consistent with the idea that both beta-arrestins are able to interact with the non-phosphorylated receptor, albeit with much lower affinity and beta-arrestin 1 less so than beta-arrestin 2. In conclusion, this study shows that the high affinity binding of beta-arrestins to the phosphorylated C-terminus is the predominant mechanism of agonist-induced beta-arrestin recruitment to the cell surface and AT1 receptor.

The IL-6-related cytokines, LIF and cardiotrophin-1, are important growth promoting and cardioprotective agents for cardiomyocytes. However, factors that regulate their actions in the heart are poorly understood. In this study, we tested the hypothesis that endothelin-1, a peptide hormone that produces a pattern of cardiac hypertrophy distinct from LIF and cardiotrophin-1, modulates LIF-induced signaling in cardiomyocytes. Upon binding LIF or cardiotrophin-1, the LIF receptor alpha subunit (LIFRalpha) dimerizes with gp130, leading to activation of constitutively associated Jak1 proteins and LIFRalpha-gp130 tyrosine phosphorylation. We found that pretreatment of neonatal rat ventricular myocytes with endothelin-1 rapidly inhibited LIF-induced LIFRalpha tyrosine phosphorylation and Jak1 activation. This effect of endothelin-1 on LIFalpha and Jak1 was attenuated by the MEK1 inhibitor, PD98059, implicating involvement of the ERK kinases. Radioligand binding studies showed that inhibition of LIF signaling resulted from a reduction in cell surface LlFRalpha levels. Additionally, endothelin-1 was found to reduce LIF-induced STAT3 activation, as indexed by STAT3 Y705 phosphorylation. Finally, endothelin-1 and LIF were shown to induce opposite patterns of STAT3 activation in cardiomyocytes. LIF induced rapid, robust STAT3 Y705 phosphorylation; endothelin-1 produced a delayed, modest increase, and initially decreased STAT3 Y705 phosphorylation. Overall our findings indicate that endothelin-1 acts to temper IL-6-related cytokine signaling in cardiomyocytes, in particular STAT3 activation.

Recent studies have shown that the JAK-STAT signaling pathway plays a central role in cardiac pathophysiology. JAK-STAT signaling has been implicated in pressure overload-induced cardiac hypertrophy and remodeling, ischemic preconditioning, and ischemia/reperfusion-induced cardiac dysfunction. The different STAT family members expressed in cardiac myocytes appear to be linked to different, and at times, opposite responses, such as cell growth/survival and apoptosis. Thus, differential activation and/or selective inhibition of the STAT proteins by agonists for G-protein coupled receptors, such as angiotensin II, may contribute to cardiac dysfunction during ischemia and heart failure. In addition, JAK-STAT signaling may represent one limb of an autocrine loop for angiotensin II generation, that serves to amplify the actions of angiotensin II on cardiac muscle. The purpose of this article is to provide an overview of recent findings that have been made for JAK-STAT signaling in cardiac myocytes and to highlight some unresolved issues for future investigation. The central focus of this review is on recent studies suggesting that modulation or activation of JAK-STAT signaling by ANG II has pathological consequences for heart function.

Transamidation is a post‐translational modification of proteins mediated by tissue transglutaminase II (TGase), a GTP‐binding protein, participating in signal transduction pathways as a non‐conventional G‐protein. Retinoic acid (RA), which is known to have a role in cell differentiation, is a potent activator of TGase. The activation of TGase results in increased transamidation of RhoA, which is inhibited by monodansylcadaverine (MDC; an inhibitor of transglutaminase activity) and TGaseM (a TGase mutant lacking transglutaminase activity). Transamidated RhoA functions as a constitutively active G‐protein, showing increased binding to its downstream target, RhoA‐associated kinase‐2 (ROCK‐2). Upon binding to RhoA, ROCK‐2 becomes autophosphorylated and demonstrates stimulated kinase activity. The RA‐stimulated interaction between RhoA and ROCK‐2 is blocked by MDC and TGaseM, indicating a role for transglutaminase activity in the interaction. Biochemical effects of TGase activation, coupled with the formation of stress fibers and focal adhesion complexes, are proposed to have a significant role in cell differentiation.

Abstract —The Janus kinase–signal transducers and activators of transcription (JAK-STAT) pathway is stimulated by angiotensin II (Ang II) via the type 1 receptor after acute pressure overload in the heart. The purpose of this study was to determine whether activation of the JAK-STAT pathway by Ang II is dependent on G proteins. Ang II (100 nmol/L for 120 minutes) caused formation of sis -inducing factor (SIF) complexes and tyrosine phosphorylation of STAT proteins in neonatal rat ventricular myocytes. The percentage of change in Ang II–stimulated SIF induction was not affected by pertussis toxin (PTX) or GP antagonist-2A, compounds that inhibit activation of G i and G o proteins. In contrast, GP antagonist-2A, a peptide that selectively inhibits activation of G q proteins, completely abolished Ang II–stimulated SIF induction and STAT3 tyrosine phosphorylation. Pretreatment of cardiac myocytes with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C (PLC) activity, decreased Ang II–stimulated SIF induction and STAT3 tyrosine phosphorylation in a dose-dependent manner. Chelation of intracellular Ca 2+ with BAPTA-AM did not alter Ang II–stimulated SIF induction. In contrast, pretreatment of cardiac myocytes with Ro-31-8220, a potent and specific inhibitor of protein kinase C (PKC), decreased Ang II–stimulated SIF induction in a dose-dependent manner. Ang II–stimulated SIF induction was abolished in cardiac myocytes after downregulation of PKC by treatment with PMA. From these data, we conclude that Ang II–stimulated SIF induction and STAT3 tyrosine phosphorylation is mediated by PTX-insensitive G proteins through a G q -PLC-PKC–mediated pathway in neonatal rat ventricular myocytes.

Abstract —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.

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.

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 …

Identification and elucidation of the roles of humoral factors involved in cardiogenesis and other aspects of cardiac growth will be important for the overall understanding of how the heart develops and functions. Angiotensin (Ang) II has been demonstrated to have important in vivo and in vitro growth-related effects in the neonate and adult myocardium. However, very little is known regarding the regulation of the cardiac renin-angiotensin system (RAS) and the role of Ang II receptors at any developmental stage. Enhanced expression of RAS during fetal and neonatal development suggests that locally produced Ang II exerts autocrine and paracrine influences on cardiogenesis and cardiac growth in these stages of development. Utilization of transgenic models and specific nonpeptide Ang II receptor antagonists will be useful in determining the roles of AT1 and AT2 receptors, respectively, in the regulation of the cardiac RAS, myocardial growth, and apoptosis in the developing rat heart. These studies may provide a better understanding of the effects of Ang II receptors on cellular and molecular aspects of cardiac development and thus lead to improved therapeutic interventions for cardiovascular disease.

Publisher Summary This chapter discusses the evidence that strongly supports the role of angiotensin Ii (Ang II) in mediating cardiac responses in the normal and failing heart. A distinction between the cardiac and superimposed circulating renin-anciotensin system (RAS) system represents a major challenge for determining biological significance of the intracardiac RAS. One possible function of this humoral system is that it provides high, localized Ang II concentrations, independent from the circulating RAS that provide an autocrine or paracrine function associated with mechanical activity and cardiac growth. Given the complexity of ACE inhibitors, utilization of renin inhibitors and selective angiotensin receptor antagonists will presumably provide a better understanding of RAS-dependent and -independent effects in cardiac development and failure. Utilization of transgenic animals also represents a powerful tool that can be utilized to address roles of particular gene products in vivo . A number of technical and biological advances have greatly simplified the process of creating both transgenic and null-mutation mice.

The Signal Transducers and Activators of Transcription (STAT) proteins are a novel class of transcription factors activated by cytokines (Interleukin-6, IL-6) and growth factors (platelet-derived growth factor, PDGF). In cultured neonatal rat cardiac fibroblasts and in CHO-K1 cells expressing angiotensin (Ang) II type 1 receptors (AT1A), called T3CHO/AT1A calls, Ang II stimulated predominandy the tyrosine phosphorylation of STAT3. In these cells, Ang II also tyrosine-phosphorylated STAT1, but tyrosine phosphorylation of STAT1 was significantly lower compared with that of STAT3. Angiotensin II-mediated activation of STAT3 compared with the rapid activation by the cytokine IL-6 was delayed (maximal 2h). However, like cytokines, Ang II rapidly induced serine phosphorylation of STAT3. Using T3CHO/AT1A cells, we examined the potential reasons for the delayed tyrosine phosphorylation of STAT3 by Ang II. We tested the possibility that the delayed tyrosine phosphorylation of STAT3 was due to the induction of an inhibitory pathway, prior to stimulation. A short pretreatment of cells with Ang II transiently inhibited the rapid STAT3 tyrosine phosphorylation by IL-6, and this inhibition could be blocked by pre-exposing the cells with the AT1 antagonist EXP3174. PD98059, a specific inhibitor of MAPK kinase 1, attenuated the inhibitory effects of Ang II. These results suggest that the inhibition by Ang II is a MAPK kinase 1-dependent process. The ability of Ang II to cross-talk with IL-6 signaling suggests a modulatory role for Ang II in cytokine-induced cellular responses.

Abstract 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 α-myosin heavy chain (α-MHC) gene expression and increases in the expression of the atrial natriuretic factor (ANF), pro-α 1 (III) collagen, and transforming growth factor β 1 (TGF-β 1 ) 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 normortensive Wistar-Kyoto (WKY) rats, and to untreated SHR with and without evidence of HF. ACEI initiated prior to failure prevented the changes in α-MHC, ANF, pro-α 1 (III) collagen, and TGF-β 1 gene expression that are associated with the transition to HF. ACEI initiated after the onset of HF lowered levels of TGF-β 1 mRNA by 50% ( P P 1 , pro-α 1 (III) collagen, and renin-angiotensin system components.

Background Diabetes-induced organ damage is significantly associated with the activation of the renin-angiotensin system (RAS). Recently, several studies have demonstrated a change in the RAS from an extracellular to an intracellular system, in several cell types, in response to high ambient glucose levels. In cardiac myocytes, intracellular angiotensin (ANG) II synthesis and actions are ACE and AT1 independent, respectively. However, a role of this system in diabetes-induced organ damage is not clear. Methods To determine a role of the intracellular ANG II in diabetic cardiomyopathy, we induced diabetes using streptozotocin in AT1a receptor deficient (AT1a-KO) mice to exclude any effects of extracellular ANG II. Further, diabetic animals were treated with a renin inhibitor aliskiren, an ACE inhibitor benazeprilat, and an AT1 receptor blocker valsartan. Results AT1a-KO mice developed significant diastolic and systolic dysfunction following 10 wks of diabetes, as determined by echocardiography. All three drugs prevented the development of cardiac dysfunction in these animals, without affecting blood pressure or glucose levels. A significant down regulation of components of the kallikrein-kinin system (KKS) was observed in diabetic animals, which was largely prevented by benazeprilat and valsartan, while aliskiren normalized kininogen expression. Conclusions These data indicated that the AT1a receptor, thus extracellular ANG II, are not required for the development of diabetic cardiomyopathy. The KKS might contribute to the beneficial effects of benazeprilat and valsartan in diabetic cardiomyopathy. A role of intracellular ANG II is suggested by the inhibitory effects of aliskiren, which needs confirmation in future studies.

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 μmol/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 (AT 1 and AT 2 ) are present in near equal proportions. Cotreatment with a 10-fold molar excess of AT 2 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%). AT 2 antagonists enhanced Ang II stimulation of protein synthesis, as indexed by [ 3 H]leucine incorporation, whereas an AT 1 antagonist blocked Ang II–induced incorporation. An AT 2 antagonist also prevented Ang II–induced protein degradation. In conclusion, Ang II–induced myocyte growth is tempered because of low AT 1 levels and an antigrowth effect of AT 2 . These findings have potential clinical significance in that regression of hypertension-induced cardiac hypertrophy by AT 1 antagonists may be in part due to an unopposed antigrowth effect of Ang II mediated via AT 2 .

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.

A new technology brings the full aperture single vision pseudophakic eye's effective hyperfocal distance within the half-meter range. A modulated index IOL containing a subsurface zeroth order coherent microlenticular mosaic defined by an index gradient adds a normalizing function to the vergences or parallactic angles of incoming light rays subtended from field object points and redirects them, in the case of near-field images, to that of far-field images. Along with a scalar reduction of the IOL's linear focal range, this results in an extreme depth of field with a narrow depth of focus and avoids the focal split-up, halo, and inherent reduction in contrast of multifocal IOLs. A high microlenticular spatial frequency, which, while still retaining an anisotropic medium, results in a nearly total zeroth order propagation throughout the visible spectrum. The curved lens surfaces still provide most of the refractive power of the IOL, and the unique holographic fabrication technology is especially suitable not only for IOLs but also for contact lenses, artificial corneas, and miniature lens elements for cameras and other optical devices.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Angiotensin II has been demonstrated to be involved in the regulation of cellular growth of several tissues in response to developmental, physiological, and pathophysiological processes. Angiotensin II has been implicated in the developmental growth of the left ventricle in the neonate and remodeling of the heart following chronic hypertension and myocardial infarction. The inhibition of DNA synthesis and collagen deposition in myocardial interstitium following myocardial infarction by angiotensin converting enzyme inhibitor, suggests that angiotensin II mediates interstitial and perivascular fibrobrosis by preventing fibroblast proliferation. In the past, little attention was focused on the identity and functional roles of cardiac fibroblasts. Recent in vitro studies utilizing cultured cardiac fibroblasts demonstrate that angiotensin II, acting via the AT1 receptor, initiates intracellular signalling pathways in common with those of peptide growth factors. Below, we describe growth-related aspects of cardiac fibroblasts with respect to angiotensin II receptors, conventional and novel signal transduction systems, secretion of extracellular matrix proteins and growth factors, and localization of renin-angiotensin system components.

Angiotensin II receptors present in cardiomyocytes, nonmyocytes (predominantly fibroblasts), nerve terminals, and the heart vasculature mediate the multiple actions of angiotensin II (AII) in the heart, including modulation of normal and patho-physiological cardiac growth. Although the cellular processes that couple AII receptors (principally the AT1 subtype) to effector responses are not completely understood, recent studies have identified an array of signal transduction pathways activated by AII in cardiac cells. These include: the stimulation of phospholipase C which results in the activation of protein kinase C and the release of calcium from intracellular stores; an enhancement of phosphaditic acid formation; the coupling to soluble tyrosine kinase phosphorylation events; the initiation of the mitogen activated protein kinase (MAPK) cascade; and the induction of the STAT (Signal Transducers and Activators of Transcription) signaling pathway. It is tempting to speculate that these latter responses, which have been previously associated with growth factor signaling pathways, are involved in AII-induced cardiac growth. Interestingly, some of these novel pathways are apparently not under the same strict control imposed upon the more classical signaling pathways. Thus, while AII-induced calcium transients are rapidly (within minutes) desensitized following exposure to AII, the MAP kinase pathway is not, and activation of the STAT pathway requires hours of agonist exposure for maximal induction. These observations support an emerging picture in which the downstream signal transduction pathways of AII receptors are initiated and terminated with a distinct temporal arrangement. This organization allows appropriate rapid responses (e.g. vascular contraction) to transient AII exposure, some of which are rapidly terminated, perhaps for protective reasons, and others not. In contrast, additional responses (e.g. growth) probably require prolonged exposure to agonist.

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor subtypes; in particular the AT1. In many tissues, the presence of multiple angiotensin II receptor subtypes, together with a low number of receptors, makes it difficult to study biological responses to physiological concentrations (10(-11)-10(-9) M) of angiotensin II. Also, cultured cells show diminished angiotensin II receptor binding with respect to time in culture and passage number. To address these problems, we expressed the recombinant AT1A receptor in CHO-K1 cells. The stably transfected receptor was characterized using radioligand binding studies and functional coupling to cytosolic free calcium. Radioligand binding of [125I] angiotensin II to the angiotensin II receptor was specific, saturable, reversible and modulated by guanine nucleotides. Like the endogenous AT1A receptor, reported in a variety of tissues, the specific, noncompetitive, nonpeptide AII receptor antagonist, EXP3174, blocked binding of [125I] angiotensin II to the transfected receptor. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.9 nM with a density of 3.4 pmol/mg protein. An important feature of many of the responses to angiotensin II is the rapid desensitization that occurs following agonist occupancy and the development of tachyphylaxis. In AT1A receptor transfected CHO-K1 cells, angiotensin II (10(-9) M) stimulated a rapid increase in cytosolic free calcium that was completely desensitized within 50 sec following receptor occupancy. Agonist induced desensitization was unaffected when receptor internalization was blocked by pretreatment with concanavalin A or incubation at 4 degrees C, and no changes in AT1A receptor affinity or number were observed. Receptor desensitization was also unaffected by inhibition or activation of protein kinase C. Thus, we have established a permanent, high-level transfectant of the AT1A receptor in CHO-K1 cells and have shown that these receptors rapidly desensitize following exposure to physiological concentrations of agonist. The mechanism of rapid desensitization is not related to receptor sequestration, internalization or controlled by PKC phosphorylation. This provides an excellent model for studying AII actions mediated through a specific receptor subtype, at subnanomolar concentrations.